JAK inhibitors containing a 4-membered heterocyclic amide

ABSTRACT

The invention provides compounds of formula (I): 
                         
which contain a 4-membered heterocyclic amide, where the variables are defined in the specification, or a pharmaceutically-acceptable salt thereof, that are useful as JAK kinase inhibitors. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat respiratory diseases, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.16/511,410, filed on Jul. 15, 2019, now allowed, which is a continuationapplication of U.S. Ser. No. 16/222,647, filed on Dec. 17, 2018, nowU.S. Pat. No. 10,392,386, which is a divisional application of U.S. Ser.No. 15/915,418, filed on Mar. 8, 2018, now U.S. Pat. No. 10,196,393,which claims the benefit of U.S. Provisional Application No. 62/469,073,filed on Mar. 9, 2017, the disclosures of which are incorporated hereinby reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The invention is directed to compounds useful as JAK kinase inhibitors.The invention is also directed to pharmaceutical compositions comprisingsuch compounds, methods of using such compounds to treat respiratorydiseases, and processes and intermediates useful for preparing suchcompounds.

State of the Art

Asthma is a chronic disease of the airways for which there are nopreventions or cures. The disease is characterized by inflammation,fibrosis, hyper-responsiveness, and remodeling of the airways, all ofwhich contribute to airflow limitation. An estimated 300 million peopleworldwide suffer from asthma and it is estimated that the number ofpeople with asthma will grow by more than 100 million by 2025. In theUnited States, asthma afflicts about 6% to 8% of the population, makingit one of the most common chronic diseases in the country. Although mostpatients can achieve control of asthma symptoms with the use of inhaledcorticosteroids that may be combined with a leukotriene modifier and/ora long acting beta agonist, there remains a subset of patients withsevere asthma whose disease is not controlled by conventional therapies.Severe persistent asthma is defined as disease that remains uncontrolledon high doses of inhaled corticosteroids. While severe asthmatics areestimated to account for approximately 5% of all asthma sufferers, theyhave a high risk of morbidity and mortality and are responsible for adisproportionate share of health care resource utilization amongasthmatics. There remains a need for novel therapies to treat thesepatients.

Cytokines are intercellular signaling molecules which includechemokines, interferons, interleukins, lymphokines, and tumor necrosisfactor. Cytokines are critical for normal cell growth andimmunoregulation but also drive immune-mediated diseases and contributeto the growth of malignant cells. Elevated levels of many cytokines havebeen implicated in the pathology of asthma inflammation. For example,antibody-based therapies targeted at interleukins (IL)-5, and 13 havebeen shown to provide clinical benefit in subsets of severe asthmapatients. Among the cytokines implicated in asthma inflammation, manyact through signaling pathways dependent upon the Janus family oftyrosine kinases (JAKs), which signal through the Signal Transducer andActivator of Transcription (STAT) family of transcription factors.Cytokines implicated in asthma inflammation which signal through theJAK-STAT pathway include IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11,IL-13, IL-23, IL-31, IL-27, thymic stromal lymphopoietin (TSLP),interferon-γ (IFNγ) and granulocyte-macrophage colony-stimulating factor(GM-CSF).

The JAK family comprises four members, JAK1, JAK2, JAK3, and tyrosinekinase 2 (TYK2). Binding of cytokine to a JAK-dependent cytokinereceptor induces receptor dimerization which results in phosphorylationof tyrosine residues on the JAK kinase, effecting JAK activation.Phosphorylated JAKs, in turn, bind and phosphorylate various STATproteins which dimerize, internalize in the cell nucleus and directlymodulate gene transcription, leading, among other effects, to thedownstream effects associated with inflammatory disease. The JAKsusually associate with cytokine receptors in pairs as homodimers orheterodimers. Specific cytokines are associated with specific JAKpairings. Each of the four members of the JAK family is implicated inthe signaling of at least one of the cytokines associated with asthmainflammation. Consequently, a chemical inhibitor with pan-activityagainst all members of the JAK family could modulate a broad range ofpro-inflammatory pathways that contribute to severe asthma.

However, the broad anti-inflammatory effect of such inhibitors couldsuppress normal immune cell function, potentially leading to increasedrisk of infection. Evidence of increased infection risk has beenobserved with the JAK inhibitor tofacitinib, which is dosed orally forthe treatment of rheumatoid arthritis. In asthma, inflammation islocalized to the respiratory tract. Inflammation of the airways ischaracteristic of other respiratory diseases in addition to asthma.Chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF),pneumonitis, interstitial lung diseases (including idiopathic pulmonaryfibrosis), acute lung injury, acute respiratory distress syndrome,bronchitis, emphysema, bronchiolitis obliterans, and sarcoidosis arealso respiratory tract diseases in which the pathophysiology is believedto be related to JAK-signaling cytokines. Local administration of a JAKinhibitor to the lungs by inhalation offers the potential to betherapeutically efficacious by delivering a potent anti-cytokine agentdirectly to the site of action, limiting systemic exposure and thereforelimiting the potential for adverse systemic immunosuppression. The needremains for a potent JAK inhibitor suitable for local administration tothe lungs for treatment of respiratory disease.

JAK-signaling cytokines also play a major role in the activation of Tcells, a sub-type of immune cells that is central to many immuneprocesses. Pathological T cell activation is critical in the etiology ofmultiple respiratory diseases. Autoreactive T cells play a role inbronchiolitis obliterans organizing pneumonia (also termed COS). Similarto COS the etiology of lung transplant rejections is linked to anaberrant T cell activation of the recipients T cells by the transplanteddonor lung. Lung transplant rejections may occur early as Primary GraftDysfunction (PGD), organizing pneumonia (OP), acute rejection (AR) orlymphocytic bronchiolitis (LB) or they may occur years after lungtransplantation as Chronic Lung Allograft Dysfunction (CLAD). CLAD waspreviously known as bronchiolitis obliterans (BO) but now is considereda syndrome that can have different pathological manifestations includingBO, restrictive CLAD (rCLAD or RAS) and neutrophilic allograftdysfunction. Chronic lung allograft dysfunction (CLAD) is a majorchallenge in long-term management of lung transplant recipients as itcauses a transplanted lung to progressively lose functionality (Gauthieret al., Curr Transplant Rep., 2016, 3(3), 185-191). CLAD is poorlyresponsive to treatment and therefore, there remains a need foreffective compounds capable of preventing or treating this condition.Several JAK-dependent cytokines such as IFNγ and IL-5 are up-regulatedin CLAD and lung transplant rejection (Berastegui et al, ClinTransplant. 2017, 31, e12898). Moreover, high lung levels of CXCR3chemokines such as CXCL9 and CXCL10 which are downstream ofJAK-dependent IFN signaling, are linked to worse outcomes in lungtransplant patients (Shino et al, PLOS One, 2017, 12 (7), e0180281).Systemic JAK inhibition has been shown to be effective in kidneytransplant rejection (Vicenti et al., American Journal ofTransplantation, 2012, 12, 2446-56). Therefore, JAK inhibitors have thepotential to be effective in treating or preventing lung transplantrejection and CLAD. Similar T cell activation events as described as thebasis for lung transplant rejection also are considered the main driverof lung graft-versus-host disease (GVHD) which can occur posthematopoietic stem cell transplants. Similar to CLAD, lung GVHD is achronic progressive condition with extremely poor outcomes and notreatments are currently approved. A retrospective, multicenter surveystudy of 95 patients with steroid-refractory acute or chronic GVHD whoreceived the systemic JAK inhibitor ruxolitinib as salvage therapydemonstrated complete or partial response to ruxolitinib in the majorityof patients including those with lung GVHD (Zeiser et al, Leukemia,2015, 29, 10, 2062-68). As systemic JAK inhibition is associated withserious adverse events and a small therapeutic index, the need remainsfor an inhaled lung-directed, non-systemic JAK inhibitor to preventand/or treat lung transplant rejection or lung GVHD.

SUMMARY OF THE INVENTION

In one aspect, the invention provides novel compounds having activity asJAK kinase inhibitors.

Accordingly, the invention provides a compound of formula (I):

wherein:

-   -   R¹ is selected from hydrogen, C₁₋₃alkyl, and C₃₋₆cycloalkyl, and        X is —C(O)R² wherein    -   R² is —NR¹³R¹⁴, wherein    -   R¹³ and R¹⁴ taken together with the nitrogen atom to which they        are attached form a 4-membered heterocyclyl, wherein the        heterocyclyl is optionally substituted with —NR⁵R⁶ and R⁷,    -   R⁵ and R⁶ are independently C₁₋₃alkyl or R⁵ and R⁶ taken        together with the nitrogen atom to which they are attached form        a 5- or 6-membered heterocyclyl optionally including an oxygen        atom,    -   R⁷ is C₁₋₃alkyl, optionally substituted with a 5- or 6-membered        heterocyclyl containing one nitrogen atom,    -   or a pharmaceutically-acceptable salt thereof.

As used hereinafter, the phrase “compound of formula (I)” means acompound of formula (I) or a pharmaceutically acceptable salt thereof;i.e., this phrase means a compound of formula (I) in free base form orin a pharmaceutically acceptable salt form unless otherwise indicated.

The invention also provides a pharmaceutical composition comprising acompound of the invention and a pharmaceutically-acceptable carrier.

The invention also provides a method of treating respiratory disease, inparticular, asthma, in a mammal, the method comprising administering tothe mammal a therapeutically effective amount of a compound or of apharmaceutical composition of the invention. In separate and distinctaspects, the invention also provides synthetic processes andintermediates described herein, which are useful for preparing compoundsof the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating respiratory disease in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

Among other aspects, the invention provides JAK kinase inhibitors offormula (I), pharmaceutically-acceptable salts thereof, andintermediates for the preparation thereof. The following substituentsand values are intended to provide representative examples of variousaspects of this invention. These representative values are intended tofurther define such aspects and are not intended to exclude other valuesor limit the scope of the invention.

In a specific aspect, R¹ is selected from hydrogen, C₁₋₃alkyl, andC₃₋₆cycloalkyl.

In another specific aspect, R¹ is selected from hydrogen and C₁₋₃alkyl.In yet another specific aspect, R¹ is C₁₋₃alkyl.

Specific values of R¹ include, but are not limited to, methyl, ethyl,n-propyl, and isopropyl.

In a specific aspect, R¹ is selected from hydrogen and C₁₋₃alkyl and Xis —C(O)R² wherein R² is

wherein R⁵ and R⁶ are independently C₁₋₃alkyl or R⁵ and R⁶ takentogether form —(CH₂)₄₋₅— and R⁷ is hydrogen or C₁₋₃alkyl.

In another specific aspect, R¹ is selected from hydrogen and C₁₋₃alkyland X is —C(O)R² wherein R² is

wherein R⁵ and R⁶ are both methyl or R⁵ and R⁶ taken together form—(CH₂)₅—; and R⁷ is hydrogen or methyl.

In another aspect, the invention provides a compound of formula (II):

wherein:

-   -   R¹ is C₁₋₃alkyl;    -   R² is

-   -   wherein        -   R⁵ and R⁶ are independently C₁₋₃alkyl or R⁵ and R⁶ taken            together form —(CH₂)₄₋₅—, R⁷ is hydrogen or C₁₋₃alkyl,            or a pharmaceutically-acceptable salt thereof.

In another specific aspect, R¹ is C₁₋₃alkyl;

-   -   R² is    -   wherein        -   R⁵ and R⁶ are both methyl or R⁵ and R⁶ taken together form            —(CH₂)₅—, and R⁷ is hydrogen or methyl.

In yet another aspect, the invention provides a compound wherein thecompound is(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]pyridin-6-yl)methanone,or a pharmaceutically-acceptable salt thereof.

In yet another aspect, the invention provides a compound wherein thecompound is(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]pyridin-6-yl)methanone.

In yet another aspect, the invention provides a compound wherein thecompound is(S)-(3-(dimethylamino)azetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]pyridin-6-yl)methanone,or a pharmaceutically-acceptable salt thereof.

In yet another aspect, the invention provides a compound wherein thecompound is(S)-(3-(dimethylamino)azetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[5,4-c]pyridin-6-yl)methanone.

In yet another aspect, the invention provides a compound selected fromthe following compounds:

-   (S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone,-   (S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone,-   (S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone,

and pharmaceutically-acceptable salts thereof.

In yet another aspect, the invention provides a compound selected fromthe following compounds:

-   (S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone,-   (S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone,    and-   (S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone.

In yet another aspect, the invention provides(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanoneof the formula

or a pharmaceutically-acceptable salt thereof.

In yet another aspect, the invention provides a compound of the formula

In one aspect, the invention provides the compounds of Examples 2, 4, 8,and Table 1 below.

Chemical structures are named herein according to IUPAC conventions asimplemented in ChemDraw software (PerkinElmer, Inc., Cambridge, Mass.).For example, the compound:

is designated as(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone.

Furthermore, the imidazo portion of the tetrahydroimidazopyridine moietyin the structure of formula (I) exists in tautomeric forms, illustratedbelow for a fragment of the compound of Example 1

According to the IUPAC convention, these representations give rise todifferent numbering of the atoms of the imidazole portion:2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine(structure A) vs.2-(1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine(structure B). It will be understood that although structures are shown,or named, in a particular form, the invention also includes the tautomerthereof.

The compounds of the invention may contain one or more chiral centersand therefore, such compounds (and intermediates thereof) can exist asracemic mixtures; pure stereoisomers (i.e., enantiomers ordiastereomers); stereoisomer-enriched mixtures and the like. Chiralcompounds shown or named herein without a defined stereochemistry at achiral center are intended to include any or all possible stereoisomervariations at the undefined stereocenter unless otherwise indicated. Thedepiction or naming of a particular stereoisomer means the indicatedstereocenter has the designated stereochemistry with the understandingthat minor amounts of other stereoisomers may also be present unlessotherwise indicated, provided that the utility of the depicted or namedcompound is not eliminated by the presence of another stereoisomer.

Compounds of formula (I) also contain several basic groups (e.g., aminogroups) and therefore, such compounds can exist as the free base or invarious salt forms, such a mono-protonated salt form, a di-protonatedsalt form, a tri-protonated salt form, or mixtures thereof. All suchforms are included within the scope of this invention, unless otherwiseindicated.

This invention also includes isotopically-labeled compounds of formula(I), i.e., compounds of formula (I) where one or more atom has beenreplaced or enriched with an atom having the same atomic number but anatomic mass different from the atomic mass that predominates in nature.Examples of isotopes that may be incorporated into a compound of formula(I) include, but are not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N,¹⁵O, ¹⁷O, and ¹⁸O. Of particular interest are compounds of formula (I)enriched in tritium or carbon-14, which compounds can be used, forexample, in tissue distribution studies. Also of particular interest arecompounds of formula (I) enriched in deuterium especially at a site ofmetabolism, which compounds are expected to have greater metabolicstability. Additionally of particular interest are compounds of formula(I) enriched in a positron emitting isotope, such as ¹¹C, ¹⁵O and ¹³N,which compounds can be used, for example, in Positron EmissionTomography (PET) studies.

Definitions

When describing this invention including its various aspects andembodiments, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl (Me),ethyl (Et), n-propyl (n-Pr) or (nPr), isopropyl (i-Pr) or (iPr), n-butyl(n-Bu) or (nBu), sec-butyl, isobutyl, tert-butyl (t-Bu) or (tBu),n-pentyl, n-hexyl, 2,2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl,2-ethylbutyl, 2,2-dimethylpentyl, 2-propylpentyl, and the like.

When a specific number of carbon atoms are intended for a particularterm, the number of carbon atoms is shown preceding the term. Forexample, the term “C₁₋₃ alkyl” means an alkyl group having from 1 to 3carbon atoms wherein the carbon atoms are in any chemically-acceptableconfiguration, including linear or branched configurations.

The term “cycloalkyl” means a monovalent saturated carbocyclic groupwhich may be monocyclic or multicyclic. Unless otherwise defined, suchcycloalkyl groups typically contain from 3 to 10 carbon atoms.Representative cycloalkyl groups include, by way of example, cyclopropyl(cPr), cyclobutyl (cBu), cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and the like.

The term “heterocyclyl”, “heterocycle”, “heterocyclic”, or “heterocyclicring” means a monovalent saturated or partially unsaturated cyclicnon-aromatic group, having from 3 to 10 total ring atoms, wherein thering contains from 2 to 9 carbon ring atoms and from 1 to 4 ringheteroatoms selected from nitrogen, oxygen, and sulfur. Heterocyclicgroups may be monocyclic or multicyclic (i.e., fused or bridged).Representative heterocyclyl groups include, by way of example,pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, morpholinyl,thiomorpholyl, indolin-3-yl, 2-imidazolinyl, tetrahydropyranyl,1,2,3,4-tetrahydroisoquinolin-2-yl, quinuclidinyl, 7-azanorbornanyl,nortropanyl, and the like, where the point of attachment is at anyavailable carbon or nitrogen ring atom. Where the context makes thepoint of attachment of the heterocyclic group evident, such groups mayalternatively be referred to as a non-valent species, i.e. pyrrolidine,piperidine, piperazine, imidazole, tetrahydropyran etc.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” means preventing, ameliorating orsuppressing the medical condition, disease or disorder being treated(e.g., a respiratory disease) in a patient (particularly a human); oralleviating the symptoms of the medical condition, disease or disorder.

The term “pharmaceutically acceptable salt” means a salt that isacceptable for administration to a patient or a mammal, such as a human(e.g., salts having acceptable mammalian safety for a given dosageregime). Representative pharmaceutically acceptable salts include saltsof acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, edisylic, fumaric, gentisic, gluconic, glucoronic,glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,lactobionic, maleic, malic, mandelic, methanesulfonic, mucic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic,pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonicand xinafoic acid, and the like.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. For example, the cation can be a protonated form ofa compound of formula (I), i.e. a form where one or more amino groupshave been protonated by an acid. Typically, the salt is apharmaceutically acceptable salt, although this is not required forsalts of intermediate compounds that are not intended for administrationto a patient.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl andtri-fluoroacetyl; alkoxycarbonyl groups, such as tert butoxycarbonyl(Boc); arylmethoxycarbonyl groups, such as benzyloxycarbonyl (Cbz) and9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups, such as benzyl(Bn), trityl (Tr), and 1,1-di-(4′-methoxyphenyl)methyl; silyl groups,such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS),[2-(trimethylsilyl)ethoxy]methyl (SEM); and the like.

The term “hydroxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a hydroxy group. Representativehydroxy-protecting groups include, but are not limited to, alkyl groups,such as methyl, ethyl, and tert-butyl; acyl groups, for example alkanoylgroups, such as acetyl; arylmethyl groups, such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); silyl groups, such as trimethylsilyl (TMS) andtert-butyldimethylsilyl (TBS); and the like.

Numerous protecting groups, and their introduction and removal, aredescribed in T. W. Greene and P.G.M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York

General Synthetic Procedures

Compounds of this invention, and intermediates thereof, can be preparedaccording to the following general methods and procedures usingcommercially-available or routinely-prepared starting materials andreagents. The substituents and variables (e.g., R¹, R², etc.) used inthe following schemes have the same meanings as those defined elsewhereherein unless otherwise indicated. Additionally, compounds having anacidic or basic atom or functional group may be used or may be producedas a salt unless otherwise indicated (in some cases, the use of a saltin a particular reaction will require conversion of the salt to anon-salt form, e.g., a free base, using routine procedures beforeconducting the reaction).

Although a particular embodiment of the present invention may be shownor described in the following procedures, those skilled in the art willrecognize that other embodiments or aspects of the present invention canalso be prepared using such procedures or by using other methods,reagents, and starting materials know to those skilled in the art. Inparticular, it will be appreciated that compounds of the invention maybe prepared by a variety of process routes in which reactants arecombined in different orders to provide different intermediates en routeto producing final products.

A general method of preparing final compounds of the invention in whichthe variable X is defined as —C(O)R² and R¹ is C₁₋₃alkyl utilizes a keyintermediate 1 and an amine of formula 2 as illustrated generally inScheme 1 and, in particular, for an example in which R² is defined as

to specifically exemplify a representative amide final product offormula (II).

To prepare amide compounds of formula (II), the carboxylic acid offormula 1 is reacted with amine 2 according to typical amide bondformation conditions. Typically, carboxylic acid 1 is contacted withbetween about 1 and about 4 equivalents of amine 2 in the presence of anexcess of base. As shown in the examples below, the amide bond formationreaction may utilize coupling agents, such asN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU) or other amide coupling agents known in theart. The reaction is typically conducted at room temperature for betweenabout 2 and about 24 hours or until the reaction is substantiallycomplete.

The carboxylic acid of formula 1 may be prepared as illustrated inScheme 2

where Pg¹ represents a hydroxy-protecting group and Pg², Pg³, and Pg⁴represent different amino-protecting groups. As described in theexamples below, a useful choice of protecting groups is benzyl or methylas Pg¹, tetrahydropyranyl (THP) as Pg², tert-butoxycarbonyl (Boc) orbenzyl as Pg³, and [2-(trimethylsilyl)ethoxylmethyl (SEM) as Pg⁴. Thefirst step of Scheme 2 is the palladium catalyzed Stille coupling ofintermediate 3 with intermediate 4 where the phenyl-indazoleintermediate 3 has the trimethylstannyl moiety and the reaction partner4 is iodine substituted. The reaction is typically conducted at elevatedtemperature, for example, at between about 80° C. and about 180° C. forbetween about 10 and about 24 hours or until the reaction issubstantially complete.

When benzyl is used as Pg¹, in the next step, the methyl ester ofintermediate 5 is converted to a benzyl ester in intermediate 6 byreaction of 5 with benzyl alcohol. Both benzyl protecting groups areconveniently removed by palladium catalyzed hydrogenation to provideintermediate 7 which may be fully deprotected by reaction with acid,typically hydrochloric acid. In a final step, the substituent IV isadded by reductive alkylation of intermediate 8 with a reagent R^(1a)where R^(1a) is an aldehyde or ketone defined such that upon reduction,R¹ is produced. For example, to add a methyl substituent R¹,formaldehyde is used as reagent R^(1a), to add an isopropyl moiety assubstituent R¹, acetone is used as reagent R^(1a). The reaction istypically conducted in the presence of a reducing agent such as sodiumcyanoborohydride or sodium triacetoxyborohydride or the like at ambienttemperature for a period of about 10 to about 24 hours or until thereaction is substantially complete.

Intermediates 3 and 4 may be prepared from commercial or easily preparedstarting materials, as described in detail below. In particular, aprocess for preparing intermediate 3 in which Pg¹ is benzyl and Pg² isTHP uses the Suzuki-Miyaura coupling of compound 9 with compound 10followed by conventional reactions to add the trimethylstannyl group.

Intermediate 4 may be prepared from compound 11, which is commerciallyavailable in racemic and stereospecific forms and may also be preparedfrom histidine.

Accordingly, in a method aspect, the invention provides a process ofpreparing a compound of formula (II) or a pharmaceutically acceptablesalt thereof, the process comprising reacting a compound of formula 1with a compound of formula 2, as illustrated in Scheme 1 to provide acompound of formula (II) or a pharmaceutically acceptable salt thereof.

In a further method aspect, the invention provides a process ofpreparing a compound of formula 1, the process comprising reacting acompound of formula 8 with R^(1a) in the presence of a reducing agent,wherein R^(1a) is an aldehyde or ketone defined such that upon reductivealkylation the substituent R¹, wherein R¹ is C₁₋₃alkyl, is attached tothe compound of formula 8 to provide the compound of formula 1.

In an additional method aspect, the invention provides a process ofpreparing a compound of formula 8 the process comprising deprotecting acompound of formula 7.

In yet another aspect, the invention provides a compound of formula 1and compounds of formula 7 and 8, useful in preparing a compound offormula 1.

Pharmaceutical Compositions

The compounds of the invention and pharmaceutically-acceptable saltsthereof are typically used in the form of a pharmaceutical compositionor formulation. Such pharmaceutical compositions may advantageously beadministered to a patient by inhalation. In addition, pharmaceuticalcompositions may be administered by any acceptable route ofadministration including, but not limited to, oral, rectal, nasal,topical (including transdermal) and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a compound offormula (I), where, as defined above, “compound of formula (I)” means acompound of formula (I) or a pharmaceutically-acceptable salt thereof.Optionally, such pharmaceutical compositions may contain othertherapeutic and/or formulating agents if desired. When discussingcompositions and uses thereof, the “compound of the invention” may alsobe referred to herein as the “active agent”. As used herein, the term“compound of the invention” is intended to include all compoundsencompassed by formula (I) as well as the species embodied in formula(II) and pharmaceutically-acceptable salts thereof.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present invention.Those skilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount.

Typically, such pharmaceutical compositions will contain from about 0.01to about 95% by weight of the active agent; including, for example, fromabout 0.05 to about 30% by weight; and from about 0.1% to about 10% byweight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the carriers or excipients used inthe pharmaceutical compositions of this invention arecommercially-available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20th Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7th Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with apharmaceutically-acceptable carrier and one or more optionalingredients. The resulting uniformly blended mixture can then be shapedor loaded into tablets, capsules, pills and the like using conventionalprocedures and equipment.

In one aspect, the pharmaceutical composition is suitable for inhaledadministration. Pharmaceutical compositions for inhaled administrationare typically in the form of an aerosol or a powder. Such compositionsare generally administered using inhaler delivery devices, such as a drypowder inhaler (DPI), a metered-dose inhaler (MDI), a nebulizer inhaler,or a similar delivery device.

In a particular embodiment, the pharmaceutical composition isadministered by inhalation using a dry powder inhaler. Such dry powderinhalers typically administer the pharmaceutical composition as afree-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free-flowing powder composition, thetherapeutic agent is typically formulated with a suitable excipient suchas lactose, starch, mannitol, dextrose, polylactic acid (PLA),polylactide-co-glycolide (PLGA) or combinations thereof. Typically, thetherapeutic agent is micronized and combined with a suitable carrier toform a composition suitable for inhalation.

A representative pharmaceutical composition for use in a dry powderinhaler comprises lactose and a compound of the invention in micronizedform. Such a dry powder composition can be made, for example, bycombining dry milled lactose with the therapeutic agent and then dryblending the components. The composition is then typically loaded into adry powder dispenser, or into inhalation cartridges or capsules for usewith a dry powder delivery device.

Dry powder inhaler delivery devices suitable for administeringtherapeutic agents by inhalation are described in the art and examplesof such devices are commercially available. For example, representativedry powder inhaler delivery devices or products include Aeolizer(Novartis); Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler(GlaxoSmithKline); Diskus/Accuhaler (GlaxoSmithKline); Ellipta(GlaxoSmithKline); Easyhaler (Orion Pharma); Eclipse (Aventis); FlowCaps(Hovione); Handihaler (Boehringer Ingelheim); Pulvinal (Chiesi);Rotahaler (GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma);Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler(Aventis); and the like.

In another particular embodiment, the pharmaceutical composition isadministered by inhalation using a metered-dose inhaler. Suchmetered-dose inhalers typically discharge a measured amount of atherapeutic agent using a compressed propellant gas. Accordingly,pharmaceutical compositions administered using a metered-dose inhalertypically comprise a solution or suspension of the therapeutic agent ina liquefied propellant. Any suitable liquefied propellant may beemployed including hydrofluoroalkanes (HFAs), such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227); and chlorofluorocarbons,such as CCl₃F. In a particular embodiment, the propellant ishydrofluoroalkanes. In some embodiments, the hydrofluoroalkaneformulation contains a co-solvent, such as ethanol or pentane, and/or asurfactant, such as sorbitan trioleate, oleic acid, lecithin, andglycerin.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a compoundof the invention; from about 0% to about 20% by weight ethanol; and fromabout 0% to about 5% by weight surfactant; with the remainder being anHFA propellant. Such compositions are typically prepared by addingchilled or pressurized hydrofluoroalkane to a suitable containercontaining the therapeutic agent, ethanol (if present) and thesurfactant (if present). To prepare a suspension, the therapeutic agentis micronized and then combined with the propellant. The composition isthen loaded into an aerosol canister, which typically forms a portion ofa metered-dose inhaler device.

Metered-dose inhaler devices suitable for administering therapeuticagents by inhalation are described in the art and examples of suchdevices are commercially available. For example, representativemetered-dose inhaler devices or products include AeroBid Inhaler System(Forest Pharmaceuticals); Atrovent Inhalation Aerosol (BoehringerIngelheim); Flovent (GlaxoSmithKline); Maxair Inhaler (3M); ProventilInhaler (Schering); Serevent Inhalation Aerosol (GlaxoSmithKline); andthe like.

In another particular aspect, the pharmaceutical composition isadministered by inhalation using a nebulizer inhaler. Such nebulizerdevices typically produce a stream of high velocity air that causes thepharmaceutical composition to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the therapeutic agent can be dissolved in a suitablecarrier to form a solution. Alternatively, the therapeutic agent can bemicronized or nanomilled and combined with a suitable carrier to form asuspension.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises a solution or suspension comprising from about 0.05μg/mL to about 20 mg/mL of a compound of the invention and excipientscompatible with nebulized formulations. In one embodiment, the solutionhas a pH of about 3 to about 8.

Nebulizer devices suitable for administering therapeutic agents byinhalation are described in the art and examples of such devices arecommercially available. For example, representative nebulizer devices orproducts include the Respimat Softmist Inhalaler (Boehringer Ingelheim);the AERx Pulmonary Delivery System (Aradigm Corp.); the PARI LC PlusReusable Nebulizer (Pari GmbH); and the like.

In yet another aspect, the pharmaceutical compositions of the inventionmay alternatively be prepared in a dosage form intended for oraladministration. Suitable pharmaceutical compositions for oraladministration may be in the form of capsules, tablets, pills, lozenges,cachets, dragees, powders, granules; or as a solution or a suspension inan aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oilliquid emulsion; or as an elixir or syrup; and the like; each containinga predetermined amount of a compound of the present invention as anactive ingredient.

When intended for oral administration in a solid dosage form, thepharmaceutical compositions of the invention will typically comprise theactive agent and one or more pharmaceutically-acceptable carriers, suchas sodium citrate or dicalcium phosphate. Optionally or alternatively,such solid dosage forms may also comprise: fillers or extenders,binders, humectants, solution retarding agents, absorption accelerators,wetting agents, absorbents, lubricants, coloring agents, and bufferingagents. Release agents, wetting agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the pharmaceutical compositions of the invention.

Alternative formulations may also include controlled releaseformulations, liquid dosage forms for oral administration, transdermalpatches, and parenteral formulations. Conventional excipients andmethods of preparation of such alternative formulations are described,for example, in the reference by Remington, supra.

The following non-limiting examples illustrate representativepharmaceutical compositions of the present invention.

Dry Powder Composition

A micronized compound of formula (I) (1 g) is blended with milledlactose (25 g). This blended mixture is then loaded into individualblisters of a peelable blister pack in an amount sufficient to providebetween about 0.1 mg to about 4 mg of the compound of formula I perdose. The contents of the blisters are administered using a dry powderinhaler.

Dry Powder Composition

A micronized compound of formula (I) (1 g) is blended with milledlactose (20 g) to form a bulk composition having a weight ratio ofcompound to milled lactose of 1:20. The blended composition is packedinto a dry powder inhalation device capable of delivering between about0.1 mg to about 4 mg of the compound of formula I per dose.

Metered-Dose Inhaler Composition

A micronized compound of formula (I) (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded intometered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 0.1mg to about 4 mg of the compound of formula I per dose when administeredby the metered dose inhaler.

Nebulizer Composition

A compound of formula (I) (25 mg) is dissolved in a solution containing1.5-2.5 equivalents of hydrochloric acid, followed by addition of sodiumhydroxide to adjust the pH to 3.5 to 5.5 and 3% by weight of glycerol.The solution is stirred well until all the components are dissolved. Thesolution is administered using a nebulizer device that provides about0.1 mg to about 4 mg of the compound of formula I per dose.

Utility

The JAK inhibitors of the invention have been designed for the treatmentof inflammatory and fibrotic disease of the respiratory tract. Inparticular, the compounds have been designed to enable delivery of apotent anti-cytokine agent directly to the site of action of respiratorydisease in the lung while limiting systemic exposure.

The compounds of the invention have been shown to be potent inhibitorsof the JAK family of enzymes: JAK1, JAK2, JAK3, and TYK2. In addition,the compounds have demonstrated potent inhibition of pro-inflammatoryand pro-fibrotic cytokines without exhibiting cytotoxicity in cellularassays. It has been recognized that the broad anti-inflammatory effectof JAK inhibitors could suppress normal immune cell function,potentially leading to increased risk of infection. The presentcompounds have therefore been optimized to limit absorption from thelung into the plasma, thus minimizing the risk of immunosuppression.

As described in the experimental section below, the absorption anddistribution of typical compounds has been profiled in preclinicalassays. Selected compounds tested in mice showed, at the same time, highconcentration in lung tissue and low absorption into plasma. Compoundstested in mouse exhibited exposure in lung from one to two orders ofmagnitude greater than exposure in plasma. The compounds also exhibitedsignificant retention in the mouse lung as evidenced by a lung half-lifegreater than about 5 hours. Importantly, the concentration of testcompound in the mouse lung has been shown to correlate with a predictedpharmacodynamic effect of JAK enzyme inhibition. Compounds of theinvention have been shown to inhibit an effect of the pro-inflammatorycytokine IL-13 in mouse lung tissue. Specifically, the compounds havedemonstrated dose and concentration dependent inhibition ofIL-13-induced phosphorylation of STAT6 in lung tissue which providesevidence of local lung JAK target engagement in vivo. This effect hasbeen observed when the pro-inflammatory cytokine IL-13 is administered 4hours after administration of the test compound, providing furtherevidence of significant retention in the lung.

Tested compounds have been demonstrated to exhibit both potentinhibitory activity at the cellular level and significant retention inlung tissue. Extensive investigation by the present inventors hasdetermined that while it is possible to identify compounds that arepotent at the cellular level or compounds that show significantretention in the lung, it is far more difficult to discover compoundsthat exhibit both desirable characteristics at the same time.

The anti-inflammatory activity of JAK inhibitors has been robustlydemonstrated in preclinical models of asthma (Malaviya et al., IntImmunopharmacol, 2010, 10, 829,-836; Matsunaga et al., Biochem andBiophys Res Commun, 2011, 404, 261-267; Kudlacz et al., Eur J Pharmacol,2008, 582, 154-161). Accordingly, the compounds of the invention areexpected to be useful for the treatment of inflammatory respiratorydisorders, in particular, asthma. Inflammation and fibrosis of the lungis characteristic of other respiratory diseases in addition to asthmasuch as chronic obstructive pulmonary disease (COPD), cystic fibrosis(CF), pneumonitis, interstitial lung diseases (including idiopathicpulmonary fibrosis), acute lung injury, acute respiratory distresssyndrome, bronchitis, emphysema, bronchiolitis obliterans, andsarcoidosis. The present compounds, therefore, are also expected to beuseful for the treatment of chronic obstructive pulmonary disease,cystic fibrosis, pneumonitis, interstitial lung diseases (includingidiopathic pulmonary fibrosis), acute lung injury, acute respiratorydistress syndrome, bronchitis, emphysema, bronchiolitis obliterans, andsarcoidosis.

The compounds of the disclosure have demonstrated inhibition of human Tcell activation, inhibition of cytokines associated with inflammation,and activity on human eosinophils and in rodent lung eosinophiliamodels. Therefore, the compounds of the disclosure are likely to beuseful for the treatment of certain specific respiratory diseases.

Eosinophilic airway inflammation is a characteristic feature of diseasescollectively termed eosinophilic lung diseases (Cottin et al., Clin.Chest. Med., 2016, 37(3), 535-56). Eosinophilic diseases have beenassociated with IL-4, IL-13 and IL-5 signaling. Eosinophilic lungdiseases include infections (especially helminthic infections),drug-induced pneumonitis (induced for example by therapeutic drugs suchas antibiotics, phenytoin, or 1-tryptophan), fungal-induced pneumonitis(e.g. allergic bronchopulmonary aspergillosis), hypersensitivitypneumonitis and eosinophilic granulomatosis with polyangiitis (formerlyknown as Churg-Strauss syndrome). Eosinophilic lung diseases of unknownetiology include idiopathic acute eosinophilic pneumoni, idiopathicchronic eosinophilic pneumonia, hypereosinophilic syndrome, and Lofflersyndrome. The compounds of the disclosure have been shown tosignificantly reduce lung eosinophilia in the rodent airway model and topotently inhibit IL-13, IL-4, and IL-2 signaling in cellular assays. Inaddition, the compound of example 2 has been demonstrated to potentlyinhibit IL-5 mediated human eosinophil survival.

A polymorphism in the IL-6 gene has been associated with elevated IL-6levels and an increased risk of developing pulmonary arterialhypertension (PAH) (Fang et al., J Am Soc Hypertens., 2017, 11(3),171-177). Corroborating the role of IL-6 in PAH, inhibition of the IL-6receptor chain gp130 ameliorated the disease in a rat model of PAH(Huang et al., Can J Cardiol., 2016, 32(11), 1356.e1-1356.e10). Thecompound of example 2 has been shown to inhibit IL-6 signaling.

Cytokines such as IFNγ, IL-12 and IL-6 have been implicated in a rangeof non-allergic lung diseases such as sarcoidosis, andlymphangioleiomyomatosis (El-Hashemite et al., Am. J. Respir. Cell Mol.Biol., 2005, 33, 227-230, and El-Hashemite et al., Cancer Res., 2004,64, 3436-3443). The compound of example 2 has also been shown to inhibitIL-6 and IFNγ signaling.

Bronchiectasis and infiltrative pulmonary diseases are diseasesassociated with chronic neutrophilic inflammation. The compound ofexample 2 has been shown to inhibit cytokines that are associated withneutrophilic inflammation (e.g. IL-6, IFNγ).

Pathological T cell activation is critical in the etiology of multiplerespiratory diseases. Autoreactive T cells play a role in bronchiolitisobliterans organizing pneumonia (also termed COS). Similar to COS theetiology of lung transplant rejections is linked to an aberrant T cellactivation of the recipients T cells by the transplanted donor lung.Lung transplant rejections may occur early as Primary Graft Dysfunction(PGD), organizing pneumonia (OP), acute rejection (AR) or lymphocyticbronchiolitis (LB) or they may occur years after lung transplantation asChronic Lung Allograft Dysfunction (CLAD). CLAD was previously known asbronchiolitis obliterans (BO) but now is considered a syndrome that canhave different pathological manifestations including BO, restrictiveCLAD (rCLAD or RAS) and neutrophilic allograft dysfunction. Chronic lungallograft dysfunction (CLAD) is a major challenge in long-termmanagement of lung transplant recipients as it causes a transplantedlung to progressively lose functionality (Gauthier et al., CurrTransplant Rep., 2016, 3(3), 185-191). CLAD is poorly responsive totreatment and therefore, there remains a need for effective compoundscapable of preventing or treating this condition. Several JAK-dependentcytokines such as IFNγ and IL-5 are up-regulated in CLAD and lungtransplant rejection (Berastegui et al, Clin Transplant. 2017, 31,e12898). Moreover, high lung levels of CXCR3 chemokines such as CXCL9and CXCL10 which are downstream of JAK-dependent IFN signaling, arelinked to worse outcomes in lung transplant patients (Shino et al, PLOSOne, 2017, 12 (7), e0180281). Systemic JAK inhibition has been shown tobe effective in kidney transplant rejection (Vicenti et al., AmericanJournal of Transplantation, 2012, 12, 2446-56). Therefore, JAKinhibitors have the potential to be effective in treating or preventinglung transplant rejection and CLAD. Similar T cell activation events asdescribed as the basis for lung transplant rejection also are consideredthe main driver of lung graft-versus-host disease (GVHD) which can occurpost hematopoietic stem cell transplants. Similar to CLAD, lung GVHD isa chronic progressive condition with extremely poor outcomes and notreatments are currently approved. A retrospective, multicenter surveystudy of 95 patients with steroid-refractory acute or chronic GVHD whoreceived the systemic JAK inhibitor ruxolitinib as salvage therapydemonstrated complete or partial response to ruxolitinib in the majorityof patients including those with lung GVHD (Zeiser et al, Leukemia,2015, 29, 10, 2062-68). As systemic JAK inhibition is associated withserious adverse events and a small therapeutic index, the need remainsfor an inhaled lung-directed, non-systemic JAK inhibitor to preventand/or treat lung transplant rejection or lung GVHD. The compounds ofthe disclosure have the characteristics required to meet this need. Morerecently, immune-checkpoint inhibitor induced pneumonitis, another Tcell mediated lung disease emerged with the increased use ofimmune-checkpoint inhibitors. In cancer patients treated with these Tcell stimulating agents, fatal pneumonitis can develop. The compound ofexample 2 has been shown to inhibit the anti-CD3 and IL-2 inducedrelease of IFNγ from activated human peripheral blood-isolated T cellsand the production of CXCL9 and CXCL10 in airway epithelial cells andthus has the potential to present a novel treatment for theseunderserved serious respiratory diseases.

In one aspect, therefore, the invention provides a method of treating arespiratory disease in a mammal (e.g., a human), the method comprisingadministering to the mammal a therapeutically-effective amount of acompound of the invention or of a pharmaceutical composition comprisinga pharmaceutically-acceptable carrier and a compound of the invention.

In one aspect, the respiratory disease is asthma, chronic obstructivepulmonary disease, cystic fibrosis, pneumonitis, chronic obstructivepulmonary disease (COPD), cystic fibrosis (CF), pneumonitis,interstitial lung diseases (including idiopathic pulmonary fibrosis),acute lung injury, acute respiratory distress syndrome, bronchitis,emphysema, bronchiolitis obliterans, or sarcoidosis. In another aspect,the respiratory disease is asthma or chronic obstructive pulmonarydisease.

In one aspect, the respiratory disease is a lung infection, aneosinophilic disease, a helminthic infection, pulmonary arterialhypertension, sarcoidosis, lymphangioleiomyomatosis, bronchiectasis, aninfiltrative pulmonary disease, drug-induced pneumonitis, fungal inducedpneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivitypneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathicacute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia,hypereosinophilic syndrome, Loffler syndrome, bronchiolitis obliteransorganizing pneumonia, acute and chronic lung transplant rejections(including PGD, OP, LB, AR and CLAD, BO, restrictive CLAD andneutrophilic allograft dysfunction), lung graft-versus-host diseasebronchiolitis obliterans organizing pneumonia, pulmonary arterialhypertension, bronchiectasis, or immune-checkpoint-inhibitor inducedpneumonitis.

The invention further provides a method of treating asthma in a mammal,the method comprising administering to the mammal atherapeutically-effective amount of a compound of the invention or of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the invention.

When used to treat asthma, the compounds of the invention will typicallybe administered in a single daily dose or in multiple doses per day,although other forms of administration may be used. The amount of activeagent administered per dose or the total amount administered per daywill typically be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

The invention further provides a method of treating a respiratorydisease (including but not limited to the disease described herein) in amammal, the method comprising administering to the mammal atherapeutically-effective amount of a compound of the invention or of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the invention.

When used to treat a respiratory disease (including but not limited tothe disease described herein), the compounds of the invention willtypically be administered in a single daily dose or in multiple dosesper day, although other forms of administration may be used. The amountof active agent administered per dose or the total amount administeredper day will typically be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered and itsrelative activity, the age, weight, and response of the individualpatient, the severity of the patient's symptoms, and the like.

As JAK inhibitors, the compounds of the disclosure may also be usefulfor a variety of other diseases. The compounds of the disclosure may beuseful for a variety of gastrointestinal inflammatory indications thatinclude, but are not limited to, inflammatory bowel disease, ulcerativecolitis (proctosigmoiditis, pancolitis, ulcerative proctitis andleft-sided colitis), Crohn's disease, collagenous colitis, lymphocyticcolitis, Behcet's disease, celiac disease, immune checkpoint inhibitorinduced colitis, ileitis, eosinophilic esophagitis, graft versus hostdisease-related colitis, and infectious colitis. Ulcerative colitis(Reimund et al., J Clin Immunology, 1996, 16, 144-150), Crohn's disease(Woywodt et al., Eur J Gastroenterology Hepatology, 1999, 11, 267-276),collagenous colitis (Kumawat et al., Mol Immunology, 2013, 55, 355-364),lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis(Weinbrand-Goichberg et al., Immunol Res, 2013, 56, 249-260), graftversus host disease-related colitis (Coghill et al., Blood, 2001, 117,3268-3276), infectious colitis (Stallmach et al., Int J Colorectal Dis,2004, 19, 308-315), Behcet's disease (Zhou et al., Autoimmun Rev, 2012,11, 699-704), celiac disease (de Nitto et al., World J Gastroenterol,2009, 15, 4609-4614), immune checkpoint inhibitor induced colitis (e.g.,CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014,12, 191), PD-1- or PD-L1-inhibitor-induced colitis), and ileitis(Yamamoto et al., Dig Liver Dis, 2008, 40, 253-259) are characterized byelevation of certain pro-inflammatory cytokine levels. As manypro-inflammatory cytokines signal via JAK activation, compoundsdescribed in this application may be able to alleviate the inflammationand provide symptom relief. In particular, the compounds of thedisclosure may be useful for the induction and maintenance of remissionof ulcerative colitis, and for the treatment of Crohn's disease, immunecheckpoint inhibitor induced colitis, and the gastrointestinal adverseeffects in graft versus host disease. In one aspect, therefore, theinvention provides a method of treating a gastrointestinal inflammatorydisease in a mammal (e.g., a human), the method comprising administeringto the mammal a compound of the disclosure or a pharmaceuticallyacceptable salt thereof or a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the disclosure ora pharmaceutically acceptable salt thereof.

Atopic dermatitis and other inflammatory skin diseases have beenassociated with elevation of proinflammatory cytokines that rely on theJAK-STAT pathway. Therefore, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, may be beneficial in a numberof dermal inflammatory or pruritic conditions that include, but are notlimited to atopic dermatitis, alopecia areata, vitiligo, psoriasis,dermatomyositis, cutaneous T cell lymphoma (Netchiporouk et al., CellCycle. 2014; 13, 3331-3335) and subtypes (Sezary syndrome, mycosisfungoides, pagetoid reticulosis, granulomatous slack skin, lymphomatoidpapulosis, pityriasis lichenoides chronica, pityriasis lichenoides etvarioliformis acuta, CD30+ cutaneous T-cell lymphoma, secondarycutaneous CD30+ large cell lymphoma, non-mycosis fungoides CD30−cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennertlymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blasticNK-cell lymphoma), prurigo nodularis, lichen planus, primary localizedcutaneous amyloidosis, bullous pemphigoid, skin manifestations of graftversus host disease, pemphigoid, discoid lupus, granuloma annulare,lichen simplex chronicus, vulvar/scrotal/perianal pruritus, lichensclerosus, post herpetic neuralgia itch, lichen planopilaris, andfoliculitis decalvans. In particular, atopic dermatitis (Bao et al.,JAK-STAT, 2013, 2, e24137), alopecia areata (Xing et al., Nat Med. 2014,20, 1043-1049), vitiligo (Craiglow et al, JAMA Dermatol. 2015, 151,1110-1112), prurigo nodularis (Sonkoly et al., J Allergy Clin Immunol.2006, 117, 411-417), lichen planus (Welz-Kubiak et al., J Immunol Res.2015, ID:854747), primary localized cutaneous amyloidosis (Tanaka etal., Br J Dermatol. 2009, 161, 1217-1224), bullous pemphigoid (Felicianiet al., Int J Immunopathol Pharmacol. 1999, 12, 55-61), and dermalmanifestations of graft versus host disease (Okiyama et al., J InvestDermatol. 2014, 134, 992-1000) are characterized by elevation of certaincytokines that signal via JAK activation. Accordingly, compounds of thedisclosure, or a pharmaceutically acceptable salt thereof, may be ableto alleviate associated dermal inflammation or pruritus driven by thesecytokines. In particular, compounds of the disclosure, or apharmaceutically acceptable salt thereof, may be expected to be usefulfor the treatment of atopic dermatitis and other inflammatory skindiseases. In one aspect, therefore, the invention provides a method oftreating an inflammatory skin disease in a mammal (e.g., a human), themethod comprising applying a pharmaceutical composition comprising acompound of the disclosure, or a pharmaceutically acceptable saltthereof and a pharmaceutical carrier to the skin of the mammal. In oneaspect, the inflammatory skin disease is atopic dermatitis.

Many ocular diseases have been shown to be associated with elevations ofproinflammatory cytokines that rely on the JAK-STAT pathway. Thecompounds of the disclosure, or a pharmaceutically acceptable saltthereof, therefore, may be useful for the treatment of a number ofocular diseases that include, but are not limited to, uveitis, diabeticretinopathy, diabetic macular edema, dry eye disease, age-relatedmacular degeneration, and atopic keratoconjunctivitis. In particular,uveitis (Horai and Caspi, J Interferon Cytokine Res, 2011, 31, 733-744),diabetic retinopathy (Abcouwer, J Clin Cell Immunol, 2013, Suppl 1,1-12), diabetic macular edema (Sohn et al., American Journal ofOpthamology, 2011, 152, 686-694), dry eye disease (Stevenson et al, ArchOphthalmol, 2012, 130, 90-100), and age-related macular degeneration(Knickelbein et al, Int Ophthalmol Clin, 2015, 55(3), 63-78) arecharacterized by elevation of certain pro-inflammatory cytokines thatsignal via the JAK-STAT pathway. Accordingly, compounds of thedisclosure, or a pharmaceutically acceptable salt thereof, may be ableto alleviate the associated ocular inflammation and reverse diseaseprogression or provide symptom relief. In one aspect, therefore, theinvention provides a method of treating an ocular disease in a mammal,the method comprising administering a pharmaceutical compositioncomprising a compound of the disclosure or a pharmaceutically-acceptablesalt thereof and a pharmaceutical carrier to the eye of the mammal. Inone aspect, the ocular disease is uveitis, diabetic retinopathy,diabetic macular edema, dry eye disease, age-related maculardegeneration, or atopic keratoconjunctivitis. In one aspect, the methodcomprises administering the compound of the disclosure, or apharmaceutically acceptable salt thereof by intravitreal injection.Compounds of the disclosure, or a pharmaceutically acceptable saltthereof, may also be used in combination with one or more compounduseful to ocular diseases.

The compounds of the disclosure, or a pharmaceutically acceptable saltthereof, may also be useful to treat other diseases such as otherinflammatory diseases, autoimmune diseases or cancers. The compounds ofthe disclosure, or a pharmaceutically acceptable salt thereof, may beuseful to treat one or more of arthritis, rheumatoid arthritis, juvenilerheumatoid arthritis, transplant rejection, xerophthalmia, psoriaticarthritis, diabetes, insulin dependent diabetes, motor neurone disease,myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock,systemic lupus erythematosus, leukemia, chronic lymphocytic leukemia,chronic myelocytic leukemia, acute lymphoblastic leukemia, acutemyelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-celllymphoma, hepatocellular carcinoma, Hodgkins disease, breast cancer,Multiple myeloma, melanoma, non-Hodgkin lymphoma, non-small-cell lungcancer, ovarian clear cell carcinoma, ovary tumor, pancreas tumor,polycythemia vera, Sjoegrens syndrome, soft tissue sarcoma, sarcoma,splenomegaly, T-cell lymphoma, and thalassemia major.

Combination Therapy

Compounds of the disclosure or a pharmaceutically acceptable saltthereof may be used in combination with one or more agents which act bythe same mechanism or by different mechanisms to treat a disease. Thedifferent agents may be administered sequentially or simultaneously, inseparate compositions or in the same composition. Useful classes ofagents for combination therapy include, but are not limited to, a beta 2adrenoceptor agonist, a muscarinic receptor antagonist, a glucocorticoidagonist, a G-protein coupled receptor-44 antagonist, a leukotriene D4antagonist, a muscarinic M3 receptor antagonist, a histamine H1 receptorantagonist, an immunoglobulin E antagonist, a PDE 4 inhibitor, an IL-4antagonist, a muscarinic M1 receptor antagonist, a histamine receptorantagonist, an IL-13 antagonist, an IL-5 antagonist, a 5-Lipoxygenaseinhibitor, a beta adrenoceptor agonist, a CCR3 chemokine antagonist, aCFTR stimulator, an immunoglobulin modulator, an interleukin 33 ligandinhibitor, a PDE 3 inhibitor, a phosphoinositide-3 kinase deltainhibitor, a thromboxane A2 antagonist, an elastase inhibitor, a Kittyrosine kinase inhibitor, a leukotriene E4 antagonist, a leukotrieneantagonist, a PGD2 antagonist, a TNF alpha ligand inhibitor, a TNFbinding agent, a complement cascade inhibitor, an eotaxin ligandinhibitor, a glutathione reductase inhibitor, an histamine H4 receptorantagonist, an IL-6 antagonist, an IL2 gene stimulator, animmunoglobulin gamma Fc receptor IIB modulator, an interferon gammaligand, an interleukin 13 ligand inhibitor, an interleukin 17 ligandinhibitor, a L-Selectin antagonist, a leukocyte elastase inhibitor, aleukotriene C₄ antagonist, a Leukotriene C4 synthase inhibitor, amembrane copper amine oxidase inhibitor, a metalloprotease-12 inhibitor,a metalloprotease-9 inhibitor, a mite allergen modulator, a muscarinicreceptor modulator, a nicotinic acetylcholine receptor agonist, anuclear factor kappa B inhibitor, a p-Selectin antagonist, a PDE 5inhibitor, a PDGF receptor antagonist, a phosphoinositide-3 kinase gammainhibitor, a TLR-7 agonist, a TNF antagonist, an Abl tyrosine kinaseinhibitor, an acetylcholine receptor antagonist, an acidic mammalianchitinase inhibitor, an ACTH receptor agonist, an actin polymerizationmodulator, an adenosine A1 receptor antagonist, an adenylate cyclasestimulator, an adrenoceptor antagonist, an adrenocorticotrophic hormoneligand, an alcohol dehydrogenase 5 inhibitor, an alpha 1 antitrypsinstimulator, an alpha 1 proteinase inhibitor, an androgen receptormodulator, an angiotensin converting enzyme 2 stimulator, an ANPagonist, a Bcr protein inhibitor, a beta 1 adrenoceptor antagonist, abeta 2 adrenoceptor antagonist, a beta 2 adrenoceptor modulator, a betaamyloid modulator, a BMP10 gene inhibitor, a BMP15 gene inhibitor, acalcium channel inhibitor, a cathepsin G inhibitor, a CCL26 geneinhibitor, a CCR3 chemokine modulator, a CCR4 chemokine antagonist, acell adhesion molecule inhibitor, a chaperonin stimulator, a chitinaseinhibitor, a collagen I antagonist, a complement C3 inhibitor, a CSF-1antagonist, a CXCR2 chemokine antagonist, a cytokine receptor commonbeta chain modulator, a cytotoxic T-lymphocyte protein-4 stimulator, adeoxyribonuclease I stimulator, a deoxyribonuclease stimulator, adipeptidyl peptidase I inhibitor, a DNA gyrase inhibitor, a DPprostanoid receptor modulator, an E-Selectin antagonist, an EGFR familytyrosine kinase receptor inhibitor, an elastin modulator, an EndothelinET-A antagonist, an Endothelin ET-B antagonist, an epoxide hydrolaseinhibitor, a FGF3 receptor antagonist, a Fyn tyrosine kinase inhibitor,a GATA 3 transcription factor inhibitor, a Glucosylceramidase modulator,a Glutamate receptor modulator, a GM-CSF ligand inhibitor, a Guanylatecyclase stimulator, a H+K+ATPase inhibitor, an hemoglobin modulator, anHeparin agonist, an Histone deacetylase inhibitor, an Histonedeacetylase-2 stimulator, an HMG CoA reductase inhibitor, an I-kappa Bkinase beta inhibitor, an ICAM1 gene inhibitor, an IL-17 antagonist, anIL-17 receptor modulator, an IL-23 antagonist, an IL-4 receptormodulator, an Immunoglobulin G modulator, an Immunoglobulin G1 agonist,an Immunoglobulin G1 modulator, an Immunoglobulin epsilon Fc receptor IAantagonist, an Immunoglobulin gamma Fc receptor IIB antagonist, anImmunoglobulin kappa modulator, an Insulin sensitizer, an Interferonbeta ligand, an Interleukin 1 like receptor antagonist, an Interleukin18 ligand inhibitor, an Interleukin receptor 17A antagonist, anInterleukin-1 beta ligand inhibitor, an Interleukin-5 ligand inhibitor,an Interleukin-6 ligand inhibitor, a KCNA voltage-gated potassiumchannel-3 inhibitor, a Kit ligand inhibitor, a Laminin-5 agonist, aLeukotriene CysLT1 receptor antagonist, a Leukotriene CysLT2 receptorantagonist, a LOXL2 gene inhibitor, a Lyn tyrosine kinase inhibitor, aMARCKS protein inhibitor, a MDR associated protein 4 inhibitor, aMetalloprotease-2 modulator, a Metalloprotease-9 modulator, aMineralocorticoid receptor antagonist, a Muscarinic M2 receptorantagonist, a Muscarinic M4 receptor antagonist, a Muscarinic M5receptor antagonist, a Natriuretic peptide receptor A agonist, a Naturalkiller cell receptor modulator, a Nicotinic ACh receptor alpha 7 subunitstimulator, a NK cell receptor modulator, a Nuclear factor kappa Bmodulator, an opioid growth factor receptor agonist, a P-Glycoproteininhibitor, a P2X3 purinoceptor antagonist, a p38 MAP kinase inhibitor, aPeptidase 1 modulator, a phospholipase A2 inhibitor, a phospholipase Cinhibitor, a plasminogen activator inhibitor 1 inhibitor, a plateletactivating factor receptor antagonist, a PPAR gamma agonist, aprostacyclin agonist, a protein tyrosine kinase inhibitor, a SH2 domaininositol phosphatase 1 stimulator, a signal transduction inhibitor, asodium channel inhibitor, a STAT-3 modulator, a Stem cell antigen-1inhibitor, a superoxide dismutase modulator, a T cell surfaceglycoprotein CD28 inhibitor, a T-cell surface glycoprotein CD8inhibitor, a TGF beta agonist, a TGF beta antagonist, a thromboxanesynthetase inhibitor, a thymic stromal lymphoprotein ligand inhibitor, athymosin agonist, a thymosin beta 4 ligand, a TLR-8 agonist, a TLR-9agonist, a TLR9 gene stimulator, a Topoisomerase IV inhibitor, aTroponin I fast skeletal muscle stimulator, a Troponin T fast skeletalmuscle stimulator, a Type I IL-1 receptor antagonist, a Type II TNFreceptor modulator, an ion channel modulator, a uteroglobin stimulator,and a VIP agonist.

Specific agents that may be used in combination with the present JAKinhibitor compounds include, but are not limited to rosiptor acetate,umeclidinium bromide, secukinumab, metenkefalin acetate, tridecactideacetate, fluticasone propionate, alpha-cyclodextrin-stabilizedsulforaphane, tezepelumab, mometasone furoate, BI-1467335, dupilumab,aclidinium, formoterol, AZD-1419, HI-1640V, rivipansel, CMP-001,mannitol, ANB-020, omalizumab, tregalizumab, Mitizax, benralizumab,golimumab, roflumilast, imatinib, REGN-3500, masitinib, apremilast,RPL-554, Actimmune, adalimumab, rupatadine, parogrelil, MK-1029,beclometasone dipropionate, formoterol fumarate, mogamulizumab,seratrodast, UCB-4144, nemiralisib, CK-2127107, fevipiprant, danirixin,bosentan, abatacept, EC-18, duvelisib, dociparstat, ciprofloxacin,salbutamol HFA, erdosteine, PrEP-001, nedocromil, CDX-0158, salbutamol,enobosarm, R-TPR-022, lenzilumab, fluticasone furoate, vilanteroltrifenatate, fluticasone propionate, salmeterol, PT-007, PRS-060,remestemcel-L, citrulline, RPC-4046, nitric oxide, DS-102, gerilimzumab,Actair, fluticasone furoate, umeclidinium, vilanterol, AG-NPP709,Gamunex, infliximab, Ampion, acumapimod, canakinumab, INS-1007, CYP-001,sirukumab, fluticasone propionate, mepolizumab, pitavastatin,solithromycin, etanercept, ivacaftor, anakinra, MPC-300-IV,glycopyrronium bromide, aclidinium bromide, FP-025, risankizumab,glycopyrronium, formoterol fumarate, Adipocell, YPL-001, tiotropiumbromide, glycopyrronium bromide, indacaterol maleate, andecaliximab,olodaterol, esomeprazole, dust mite vaccine, mugwort pollen allergenvaccine, vamorolone, gefapixant, revefenacin, gefitinib, Rejoin,tipelukast, bedoradrine, SCM-CGH, SHP-652, RNS-60, brodalumab,BIO-11006, umeclidinium bromide, vilanterol trifenatate, ipratropiumbromide, tralokinumab, PUR-1800, VX-561, VX-371, olopatadine,tulobuterol, formoterol fumarate, triamcinolone acetonide, reslizumab,salmeterol xinafoate, fluticasone propionate, beclometasonedipropionate, formoterol fumarate, tiotropium bromide, ligelizumab,RUTI, bertilimumab, omalizumab, glycopyrronium bromide, SENS-111,beclomethasone dipropionate, CHF-5992, LT-4001, indacaterol,glycopyrronium bromide, mometasone furoate, fexofenadine, glycopyrroniumbromide, azithromycin, AZD-7594, formoterol, CHF-6001, batefenterol,OATD-01, olodaterol, CJM-112, rosiglitazone, salmeterol, setipiprant,inhaled interferon beta, AZD-8871, plecanatide, fluticasone, salmeterol,eicosapentaenoic acid monoglycerides, lebrikizumab, RG-6149, QBKPN,Mometasone, indacaterol, AZD-9898, sodium pyruvate, zileuton, CG-201,imidafenacin, CNTO-6785, CLBS-03, mometasone, RGN-137, procaterol,formoterol, CCI-15106, POL-6014, indacaterol, beclomethasone, MV-130,GC-1112, Allergovac depot, MEDI-3506, QBW-251, ZPL-389, udenafil,GSK-3772847, levocetirizine, AXP-1275, ADC-3680, timapiprant,abediterol, AZD-7594, ipratropium bromide, salbutamol sulfate, tadekinigalfa, ACT-774312, dornase alfa, iloprost, batefenterol, fluticasonefuroate, alicaforsen, ciclesonide, emeramide, arformoterol, SB-010,Ozagrel, BTT-1023, Dectrekumab, levalbuterol, pranlukast, hyaluronicacid, GSK-2292767, Formoterol, NOV-14, Lucinactant, salbutamol,prednisolone, ebastine, dexamethasone cipecilate, GSK-2586881,BI-443651, GSK-2256294, VR-179, VR-096, hdm-ASIT+, budesonide,GSK-2245035, VTX-1463, Emedastine, dexpramipexole, levalbuterol, N-6022,dexamethasone sodium phosphate, PIN-201104, OPK-0018, TEV-48107,suplatast, BI-1060469, Gemilukast, interferon gamma, dalazatide,bilastine, fluticasone propionate, salmeterol xinafoate, RP-3128,bencycloquidium bromide, reslizumab, PBF-680, CRTH2 antagonist,Pranlukast, salmeterol xinafoate, fluticasone propionate, tiotropiumbromide monohydrate, masilukast, RG-7990, Doxofylline, abediterol,glycopyrronium bromide, TEV-46017, ASM-024, fluticasone propionate,glycopyrronium bromide, salmeterol xinafoate, salbutamol, TA-270,Flunisolide, sodium chromoglycate, Epsi-gam, ZPL-521, salbutamol,aviptadil, TRN-157, Zafirlukast, Stempeucel, pemirolast sodium, nadolol,fluticasone propionate+salmeterol xinafoate, RV-1729, salbutamolsulfate, carbon dioxide+perfluorooctyl bromide, APL-1,dectrekumab+VAK-694, lysine acetylsalicylate, zileuton, TR-4, humanallogenic adipose-derived mesenchymal progenitor cell therapy,MEDI-9314, PL-3994, HMP-301, TD-5471, NKTT-120, pemirolast,beclomethasone dipropionate, trantinterol, monosodium alpha luminol,IMD-1041, AM-211, TBS-5, ARRY-502, seratrodast, recombinant midismase,ASM-8, deflazacort, bambuterol, RBx-10017609, ipratropium+fenoterol,fluticasone+formoterol, epinastine, WIN-901X,VALERGEN-DS,OligoG-COPD-5/20, tulobuterol, oxis Turbuhaler, DSP-3025,ASM-024, mizolastine, budesonide+salmeterol, LH-011, AXP-E, histaminehuman immunoglobulin, YHD-001, theophylline, ambroxol+erdosteine,ramatroban, montelukast, pranlukast, AG-1321001, tulobuterol,ipratropium+salbutamol, tranilast, methylprednisolone suleptanate,colforsin daropate, repirinast, and doxofylline.

Also provided, herein, is a pharmaceutical composition comprising acompound of the disclosure or a pharmaceutically acceptable salt thereofand one or more other therapeutic agents. The therapeutic agent may beselected from the class of agents specified above and from the list ofspecific agent described above. In some embodiments, the pharmaceuticalcomposition is suitable for delivery to the lungs. In some embodiments,the pharmaceutical composition is suitable for inhaled or nebulizedadministration. In some embodiments, the pharmaceutical composition is adry powder or a liquid composition.

Further, in a method aspect, the invention provides a method of treatinga disease or disorder in a mammal comprising administering to the mammala compound of the disclosure or a pharmaceutically acceptable saltthereof and one or more other therapeutic agents.

When used in combination therapy, the agents may be formulated in asingle pharmaceutical composition, or the agents may be provided inseparate compositions that are administered simultaneously or atseparate times, by the same or by different routes of administration.Such compositions can be packaged separately or may be packaged togetheras a kit. The two or more therapeutic agents in the kit may beadministered by the same route of administration or by different routesof administration.

Compounds of the invention have been demonstrated to be potentinhibitors of the JAK1, JAK2, JAK3, and TYK2 enzymes in enzyme bindingassays, to have potent functional activity without cytotoxicity incellular assays, and to exert the pharmacodynamic effects of JAKinhibition in preclinical models, as described in the followingexamples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   ACN=acetonitrile    -   DCM=dichloromethane    -   DIPEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   EtOAc=ethyl acetate    -   h=hour(s)    -   HATU=N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   IPA=isopropyl alcohol    -   IPAc=isopropylacetate    -   MeOH=methanol    -   min=minute(s)    -   Pd(PPh₃)₄=tetrakis(triphenylphosphine)palladium(0)    -   RT=room temperature    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran    -   bis(pinacolato)diboron=4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl]

Reagents and solvents were purchased from commercial suppliers (Aldrich,Fluka, Sigma, etc.), and used without further purification. Progress ofreaction mixtures was monitored by thin layer chromatography (TLC),analytical high performance liquid chromatography (anal. HPLC), and massspectrometry. Reaction mixtures were worked up as described specificallyin each reaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by column chromatography or by preparative HPLC,typically using C18 or BDS column packings and conventional eluents.Typical preparative HPLC conditions are described below.

Characterization of reaction products was routinely carried out by massand ¹H-NMR spectrometry. For NMR analysis, samples were dissolved indeuterated solvent (such as CD₃OD, CDCl₃, or d₆-DMSO), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (400 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with an Applied Biosystems (Foster City, Calif.) model API 150 EXinstrument or a Waters (Milford, Mass.) 3100 instrument, coupled toautopurification systems.

Preparative HPLC Conditions

-   Column: C18, 5 μm. 21.2×150 mm or C18, 5 μm 21×250 or C14, 5 μm    21×150 mm-   Column temperature: Room Temperature-   Flow rate: 20.0 mL/min-   Mobile Phases: A=Water+0.05% TFA    -   B=ACN+0.05% TFA,-   Injection volume: (100-1500 μL)-   Detector wavelength: 214 nm

Crude compounds were dissolved in 1:1 water:acetic acid at about 50mg/mL. A 4 minute analytical scale test run was carried out using a2.1×50 mm C₁₈ column followed by a 15 or 20 minute preparative scale runusing 100 μL injection with the gradient based on the % B retention ofthe analytical scale test run. Exact gradients were sample dependent.Samples with close running impurities were checked with a 21×250 mm C18column and/or a 21×150 mm C14 column for best separation. Fractionscontaining desired product were identified by mass spectrometricanalysis.

Preparation 1:2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(9)

(a) 1-(Benzyloxy)-4-bromo-5-ethyl-2-fluorobenzene (21)

To a solution of 4-bromo-5-ethyl-2-fluorophenol (20) (20 g, 910.32 mmol)in ACN (250 mL) was added K₂CO₃ (31.55 g, 228.3 mmol) followed by benzylbromide (13.10 mL, 109.58 mmol) drop wise. The resulting reactionmixture was stirred at 80° C. for 2 h. The aqueous layer was extractedwith EtOAc (three times), combined and washed with brine. The organiclayer was dried over Na₂SO₄ and evaporated under reduced pressure toafford the title intermediate as a pale yellow oily liquid (25 g, 89%yield). ¹H NMR (400 MHz, chloroform-d) δ 7.48-7.30 (m, 5H), 7.27 (d,J=10.5 Hz, 1H), 6.87 (d, J=8.7 Hz, 1H), 5.12 (s, 2H), 2.66 (q, J=7.5 Hz,2H), 1.16 (t, J=7.5 Hz, 3H).

(b)2-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(9)

To a solution of the product of the previous step (21) (12.5 g, 40.45mmol) in dioxane (100 mL) was added bis(pinacolato)diboron (15.40 g,60.67 mmol) and KOAc (11.9 g, 121.35 mmol). The reaction mixture waspurged with nitrogen for 15 min followed by addition of[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complexwith dichloromethane (1.65 g, 2.023 mmol). The resulting reactionmixture was stirred and heated at 110° C. for 3 h, filtered throughCelite and the residue washed with EtOAc. The filtrate was diluted withexcess EtOAc (200 mL) and washed with water (100 mL) followed by brine(100 mL), dried over sodium sulfate and concentrated in vacuo to getcrude product which was purified by column chromatography over (100-200)silica gel, eluted with 3-5% EtOAc: hexane to afford the desired productas an off-white solid (9.50 g, 66% yield). ¹H NMR (400 MHz,chloroform-d) δ 7.54-7.27 (m, 6H), 6.81 (d, J=7.9 Hz, 1H), 5.16 (s, 2H),2.84 (q, J=7.5 Hz, 2H), 1.32 (s, 12H), 1.14 (t, J=7.5 Hz, 3H).

Preparation 2:6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-indazole(3′)

(a)6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(22)

To a solution of 6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (10)(50 g, 178.57 mmol) and2-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(9) (76.3 g, 214.29 mmol) in DMF:H₂O (480:120 mL) was added K₃PO₄ (94.64g, 446.86 mmol). The reaction mixture was degassed with nitrogen for 15min, then Pd(PPh₃)₂Cl₂ catalyst (6.26 g, 8.93 mmol) was added and themixture was again degassed with nitrogen for 5 min stirred, and heatedat 100-110° C. for 5 h. The reaction mixture was filtered through Celiteand the residue was washed with EtOAc. The filtrate was diluted withEtOAc, washed with cold water and brine, dried over sodium sulfate andconcentrated in vacuo to provide crude product which was purified byflash column chromatography to afford the title intermediate as a whitesolid (65 g, 86% yield). (m/z): [M+H]⁺ calcd for C₂₇H27FN₂O₂ 431.21found 431.46. ¹H NMR (400 MHz, chloroform-d) δ 8.06-7.98 (m, 2H), 7.70(d, J=8.2 Hz, 1H), 7.51-7.32 (m, 5H), 7.08 (dd, J=809.6, 8.3 Hz, 1H),7.03 (d, J=11.9 Hz, 1H), 6.95 (d, J=8.5 Hz, 1H), 5.76-5.64 (m, 1H), 5.20(s, 2H), 4.04 (d, J=10.1 Hz, 1H), 3.72 (t, J=9.7 Hz, 1H), 2.52 (q, J=7.5Hz, 2H), 2.22-2.02 (m, 3H), 1.80-1.71 (m, 3H), 1.06 (t, J=7.5 Hz, 3H).

(b) 6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazole (23)

To a solution of the product of the previous step (22) (65 g, 151.16mmol) in methanol (700 mL) was added conc. HCl (120 mL) and theresulting solution was heated at 60-65° C. for 3 h, cooled to RT, andconcentrated in vacuo. The residue was dissolved in EtOAc and washedwith saturated NaHCO₃ aqueous solution and water. The organic layer wasdried over anhydrous Na₂SO₄ and concentrated in vacuo to afford thetitle intermediate as a white solid (52 g, 99% (crude)). ¹H NMR (400MHz, chloroform-d) δ 8.13 (s, 1H), 7.77 (d, J=8.3 Hz, 1H), 7.59-7.30 (m,6H), 7.10 (d, J=8.3 Hz, 1H), 7.01 (d, J=11.8 Hz, 1H), 6.96 (d, J=8.4 Hz,1H), 5.21 (s, 2H), 2.53 (q, J=7.5 Hz, 2H), 1.05 (t, J=7.5 Hz, 3H).

(c) 6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-3-iodo-1H-indazole (24)

To a solution of 6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1H-indazole(23) (56 g, 161.18 mmol) in DMF (400 mL) was added KOH (36.2 g, 647.39mmol) and the mixture was stirred for 5 min. A solution of iodine (82.2g, 323.69 mmol) in DMF (100 mL) was added slowly at 0° C. and stirred atRT for 30 min, diluted with water (3×150 mL) and extracted with EtOAc(3×200 mL). The organic layer was washed with saturated sodiummetabisulfite aqueous solution (3×200 mL) and water (400 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to get crudeproduct which was purified by flash column chromatography to afford thetitle intermediate as a brownish semi-solid (64 g, 84% yield). ¹H NMR(400 MHz, chloroform-d) δ 10.49 (s, 1H), 7.57-7.32 (m, 7H), 7.16 (d,J=8.3 Hz, 1H), 7.04-6.91 (m, 2H), 5.20 (s, 2H), 2.51 (q, J=7.4 Hz, 2H),1.04 (t, J=7.5 Hz, 3H).

(d)6-(4-(Benzyloxy)-2-ethyl-5-fluorophenyl)-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(25)

To an ice-cold solution of the product of the previous step (24) (60 g,127.12 mmol) in DCM (700 mL) was added p-toluensulfonic acid (4.84 g,25.423 mmol) followed by 3,4-dihydro-2H-pyran (17.43 mL, 190.68 mmol)drop wise. The reaction mixture was stirred at RT overnight, dilutedwith DCM and washed with saturated NaHCO₃ aqueous solution and brine.The organic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to provide crude product which was purified by flashchromatography (silica gel) to afford the title intermediate as an offwhite solid (64 g, 91% yield). (m/z): [M+H]⁺ calcd for C₂₇H₂₆FIN₂O₂557.10 found 557.30. ¹H NMR (400 MHz, chloroform-d) δ 7.56-7.31 (m, 7H),7.14 (d, J=8.3 Hz, 1H), 7.01 (d, J=11.8 Hz, 1H), 6.95 (d, J=8.5 Hz, 1H),5.68 (d, J=9.3 Hz, 1H), 5.20 (s, 2H), 4.08-3.99 (m, 1H), 3.77-3.64 (m,1H), 2.50 (q, J=7.2 Hz, 2H), 2.23-1.97 (m, 3H), 1.81-1.68 (m, 3H), 1.06(t, J=7.4 Hz, 3H).

(e)6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-indazole(3′)

To a solution of6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole(25) (20 g, 35.97 mmol) in toluene (150 mL) was added hexamethylditin(9.2 mL, 43.17 mmol). The reaction mixture was degassed with nitrogenfor 20 min followed by addition of tetrakis (2.0 g, 1.80 mmol) and thenstirred at 100° C. for 2 h, cooled to RT, filtered through Celite andresidue washed with EtOAc The filtrate was concentrated and purified bycolumn chromatography (over neutral alumina), eluted with 2-5%.EtOAc:hexane to afford the title compound (17.50 g, 82% yield). (m/z):[M+H]⁺ calcd for C₂₇H₂₆FIN₂O₂ 557.10 found 557.30. (m/z): [M+H]⁺ calcdfor C₃₀H₃₅FN₂O₂Sn 595.17, 593.17 found 595.49, 593.55. ¹H NMR (400 MHz,chloroform-d) δ 7.68 (d, J=8.0 Hz, 1H), 7.57-7.29 (m, 6H), 7.13-7.00 (m,2H), 6.96 (d, J=8.4 Hz, 1H), 5.81-5.68 (m, 1H), 5.21 (s, 2H), 4.13-4.00(m, 1H), 3.81-3.66 (m, 1H), 2.54 (q, J=7.3 Hz, 2H), 2.23-2.00 (m, 2H),1.87-1.59 (m, 4H), 1.08 (t, J=7.5 Hz, 3H), 0.47 (s, 9H).

Preparation 3: 5-(tert-butyl) 6-methyl(S)-2-iodo-3-((2-trimethylsilyl)ethoxy)methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate(4′)

(a) (S)-4,5,6,7-Tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid(11)

To a stirred suspension of L-histidine (26) (50 g, 322.24 mmol) in water(420 mL) was added conc. HCl (29 mL) drop wise at 0° C. followed byformaldehyde (55 mL, 676.72 mmol) in one portion at 0° C. The resultingreaction mixture was stirred for 30 min and then heated at 75° C. for 6h and concentrated. The resulting crude was stirred for 2 h with diethylether, filtered and washed with IPA:THF (100:300 mL) to provide the HClsalt of the title intermediate as an off white solid (75 g 99% yield(crude)). (m/z): [M+H]⁺ calcd for C₇H₉N₃O₂ 168.07 found 168.17.

(b) Methyl(S)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylate (27)

To a stirred solution of the product of the previous step (11) (75.0 g,312.5 mmol) in methanol (1500 mL) was added SOCl₂ (45.6 mL, 625 mmol)dropwise at 0° C. and stirred at RT for 16 h, then heated up to reflux(70° C.) for 1 h. The solvent was removed by distillation and the crudeproduct was triturated with methanol followed by diethyl ether toprovide the crude HCl salt of the title intermediate as an off whitesolid (80 g crude). ¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (s, 1H), 4.71 (dd,J=9.4, 5.2 Hz, 1H), 4.36 (d, J=15.5 Hz, 1H), 4.30 (d, J=15.6 Hz, 1H),3.82 (s, 3H), 3.44-3.21 (m, 2H).

(c) 5-(tert-Butyl) 6-methyl(S)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate (28)

To a stirred solution of the product of the previous step (27) (80.0 g,314.96 mmol) in methanol (1000 mL) was added DIPEA (282 mL, 1574 mmol)followed by di-tert-butyl dicarbonate (172 mL, 787.48 mmol) at 0° C. Thereaction mixture was stirred at RT for 16 h and then liquid NH₃ (150 mL,25% in water) was added and the reaction mixture was stirred again for16 h at RT, methanol was removed by distillation and the residue wasextracted in DCM (3×200 mL). Combined organic extracts were dried overanhydrous Na₂SO₄, concentrated and purified by flash chromatography(100-200 mesh silica gel), eluted with 5% MeOH:DCM to afford the titleintermediate (41 g, 46%. yield). (m/z): [M+H]⁺ calcd for C₁₃H₁₉N₃O₄282.14 found 282.21. ¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 1H), 7.50 (s,1H), 5.18 (dd, J=49.3, 5.1 Hz, 1H), 4.51 (t, J=14.2 Hz, 1H), 4.09 (dd,J=43.9, 16.1 Hz, 1H), 3.59 (s, 3H), 3.08 (d, J=15.5 Hz, 1H), 2.94 (d,J=15.1 Hz, 1H), 1.45 (s, 9H).

(d) 5-(tert-Butyl) 6-methyl(S)-2-iodo-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate(29)

To a solution of the product of the previous step (29) (41.0 g, 145.9mmol) in THF (500 mL) was added N-iodosuccinimide (66.0 g, 291.8 mmol)at 0° C. and the resulting solution was stirred at RT for 4 h, dilutedwith water and extracted with ethyl acetate. The organic portion waswashed with 10% sodium thiosulphate solution (3×200 mL). The combinedorganic layer was dried over anhydrous sodium sulfate, and concentratedto provide the title compound 60 g (crude), which was used in the nextstep without further purification. (m/z): [M+H]⁺ calcd for C₁₃H₁₈IN₃O₄408.03 found 408.31. ¹H NMR (400 MHz, DMSO-d₆) δ 12.48 (s, 1H),5.34-4.97 (m, 1H), 4.67-4.35 (m, 1H), 4.12-3.95 (m, 1H), 3.60 (s, 3H),3.14-2.82 (m, 2H), 1.44 (s, 9H).

(e) 5-(tert-Butyl) 6-methyl (S)-2-iodo-3-((2-trimethylsilyl)ethoxy)methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate(4′)

To a stirred solution of 5-(tert-butyl) 6-methyl(S)-2-iodo-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate(29) (40 g, 0.098 mol) in DMF (150 mL) was added DIPEA (35.1 mL, 0.19mol) at 0° C. The reaction mixture was stirred for 10 min then2-(trimethylsilyl)-ethoxymethyl chloride (19.1 mL, 0.10 mol) was addeddrop wise at 0° C. The resulting reaction mixture was stirred for 3 h atRT. After 4 h chilled water was added and the reaction mixture wasextracted with EtOAc (2×200 mL). The organic layer was dried overanhydrous sodium sulphate, concentrated, and purified by flash columnchromatography, eluted with 20-35% EtOAc:hexane, to afford the titleproduct as a pale yellow viscous liquid (27 g). (m/z): [M+H]⁺ calcd forC₁₉H₃₂IN₃O₅Si 538.12 found 538.42. ¹H NMR (400 MHz, DMSO-d₆) δ 5.33-5.04(m, 3H), 4.79-4.56 (m, 1H), 4.54-4.14 (m, 1H), 3.60 (s, 3H), 3.47 (t,J=7.8 Hz, 2H), 3.31-3.16 (m, 1H), 2.97 (t, J=18.9 Hz, 1H), 1.44 (s, 9H),0.92-0.74 (m, 2H), −0.03 (s, 9H).

Preparation 4:(6S)-5-(tert-butoxycarbonyl)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid (7′)

(a) 5-(tert-Butyl) 6-methyl(6S)-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate(5′)

To a stirred solution of 5-(tert-butyl) 6-methyl(S)-2-iodo-3-((2-trimethylsilyl)ethoxy)methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate(4′) (17.0 g, 31.65 mmol) in toluene (500 mL) was added6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-3-(trimethylstannyl)-1H-indazole(3′) (20 g, 34.82 mmol). The reaction mixture was purged with argon for15 min, Pd(PPh₃)₄ (3.6 g, 3.16 mmol) and copper iodide (1.20 g, 6.33mmol) were added and the reaction mixture was stirred at 120° C. for 16h. The reaction mixture was filtered through Celite, the filtrate wasconcentrated under reduced pressure and purified by silica gel columnchromatography (Redisep 80 g column, eluted with DCM for 10 min and then15-20% EtOAc in Hexane to afford the title intermediate as a yellowsolid (15.10 g, 58% yield). (m/z): [M+H]⁺ calcd for C₄₆H₅₈FN₅O₇Si 840.41found 840.54. ¹H NMR (400 MHz, Chloroform-d) δ 8.43 (s, 1H), 7.54-7.33(m, 6H), 7.20 (s, 1H), 7.05 (d, J=11.4 Hz, 1H), 6.95 (d, J=8.5 Hz, 1H),6.09-5.69 (m, 3H), 5.59-5.36 (m, 1H), 5.20 (s, 2H), 4.97-4.80 (m, 1H),4.12-3.90 (m, 1H), 3.68 (s, 3H), 3.57-3.47 (m, 2H), 3.40 (d, 1H),3.21-3.05 (m, 1H), 2.74-2.34 (m, 4H), 2.25-2.07 (m, 2H), 1.94-1.65 (m,4H), 1.54 (s, 9H), 1.12-0.99 (m, 3H), 0.91-0.75 (m, 2H), −0.12 (s, 9H).

(b) 6-Benzyl 5-(tert-butyl)(6S)-2-(6-(4-(benzyloxy)-2-ethyl-5-fluorophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate(6′)

To a round bottom flask was added the product of the previous step (5′)(15.0 g, 17.85 mmol) in toluene (400 mL), benzyl alcohol (46.3 mL) andTi(OEt)₄ (7.15 mL, 35.70 mmol) and the reaction mixture was refluxedvigorously (140° C.) for 48 h, diluted with water and extracted withDCM. The suspension was filtered, filtrate was dried over Na₂SO₄,concentrated under reduced pressure and purified by silica gel columnchromatography (Redisep 80 g column, 0-5% EtOAc in hexanes) for 20 minto remove excess benzyl alcohol, then eluted with 10-15% EtOAc inHexane) to provide the title intermediate. ¹H NMR consistent withstructure. (m/z): [M+H]⁺ calcd for C₅₂H₆₂FN₅O₇Si 916.44 found 916.86.

(c)(6S)-5-(tert-butoxycarbonyl)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid (7′)

To a stirred solution of the product of the previous step (6′) (21.0 g,22.92 mmol) in 1:1 IPA:THF (400 mL)) was added Pd(OH)₂ (5.0 g). Thereaction mixture was stirred at RT for 16 h under a hydrogen balloon,filtered through Celite, concentrated under reduced pressure, andpurified by silica gel column chromatography (Redisep 80 g column,eluted with 25-40% EtOAc in Hexane) to provide the title compound (6.1g, 8.29 mmol) as an off-white solid). (m/z): [M+H]⁺ calcd forC₃₈H₅₀FN₅O₇Si 736.35 found 736.5. ¹H NMR consistent with structure.(m/z): [M+H]⁺ calcd for C38H₅₀FN₅O₇Si 736.35 found 736.5. ¹H NMR (400MHz, DMSO-d₆) δ 12.94 (s, 1H), 9.86 (s, 1H), 8.34 (t, J=7.6 Hz, 1H),7.66 (s, 1H), 7.20 (d, J=8.7 Hz, 1H), 7.03 (d, J=11.8 Hz, 1H), 6.93 (d,J=9.1 Hz, 1H), 6.11-5.77 (m, 3H), 5.33-5.06 (m, 1H), 4.87-4.56 (m, 1H),4.52-4.14 (m, 1H), 3.97-3.69 (m, 2H), 3.53-3.40 (m, 2H), 3.23-3.11 (m,1H), 3.11-2.93 (m, 1H), 2.47-2.44 (m, 2H), 2.13-1.96 (m, 2H), 1.68 (d,J=70.9 Hz, 4H), 1.48 (s, 9H), 1.02 (t, J=7.5 Hz, 3H), 0.86-0.68 (m, 2H),−0.17 (s, 9H).

Preparation 5:(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid (8′)

To a stirred solution of(6S)-5-(tert-butoxycarbonyl)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-3-yl)-3-((2-(trimethylsilyl)ethoxy)-methyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid (7′) (5.7 g, 7.75 mmol) in 5:1 dioxane:water (60 mL) was addedconc. HCl (20 mL) drop wise at 0° C. The reaction mixture was warmed andstirred at 90° C. for 16 h and distilled under vacuum to provide thecrude residue, which was sequentially triturated with chilled diethylether and acetonitrile to provide the HCl salt of the title compound(3.6 g. 95% yield) as a light brown solid. (m/z): [M+H]⁺ calcd forC₂₂H₂₀FN₅O₃ 422.16 found 422.24. ¹H NMR (400 MHz, D₂0/DMSO-d₆) δ 8.22(d, J=8.4 Hz, 1H), 7.49 (s, 1H), 7.19 (d, J=8.1 Hz, 1H), 6.99 (d, J=11.9Hz, 1H), 6.91 (d, J=9.0 Hz, 1H), 4.56-4.51 (m, 1H), 4.36 (d, J=15.5 Hz,1H), 4.30 (d, J=15.5 Hz, 1H), 3.35-3.25 (m, 1H), 3.15-3.05 (m, 1H),2.4-2.55 (m, 2H), 0.97 (t, J=7.5 Hz, 3H).

Preparation 6:(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid

To a solution of(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, HCl (400 mg, 0.874 mmol) (8′) and propionaldehyde (0.095 mL, 1.310mmol) in DMF (7 mL), was added sodium cyanoborohydride (165 mg, 2.62mmol) and the reaction mixture was stirred at RT overnight. Sodiumborohydride (33 mg, 0.874 mmol) was added, the solution wasconcentrated, and purified by preparative HPLC to provide the TFA saltof the title compound (179 mg, 37% yield). (m/z): [M+H]⁺ calcd forC₂₅H₂₆FN₅O₃ 464.20 found 464.5.

Preparation 7:(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid

To a solution of(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, HCl (8′) (400 mg, 0.874 mmol), acetone (0.192 mL, 2.62 mmol), andacetic acid (0.150 mL, 2.62 mmol) in DMF (7 mL), was added sodiumcyanoborohydride (274 mg, 4.37 mmol) and the reaction mixture wasstirred at RT overnight. Sodium borohydride (33 mg, 0.874 mmol) wasadded, the solution was concentrated, and purified by preparative HPLCto provide the TFA salt of the title compound (115 mg, 23% yield).(m/z): [M+H]⁺ calcd for C₂₅H₂₆FN₅O₃ 464.20 found 464.5.

Preparation 8:(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-methyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid

To a solution of(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, HCl (8′) (300 mg, 0.655 mmol) and 37 wt. % formaldehyde in water(0.059 mL, 0.786 mmol) DMF (5 mL), was added sodium cyanoborohydride(165 mg, 2.62 mmol) and the reaction mixture was stirred at RTovernight. Sodium borohydride 25 mg, 0.655 mmol) was added, the solutionwas concentrated, and purified by flash chromatography (100 g column,5-75% ACN/water), to provide the TFA salt of the title compound (85 mg,24% yield). (m/z): [M+H]⁺ calcd for C₂₃H₂₂FN₅O₃ 436.17 found 436.45.

Preparation 9:(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid

To a solution of(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, HCl (8′) (450 mg, 0.983 mmol) and acetaldehyde (0.083 mL, 1.474mmol) in DMF (7 mL), was added sodium cyanoborohydride (247 mg, 3.93mmol) and the reaction mixture was stirred at RT overnight. Sodiumborohydride (112 mg, 2.95 mmol) was added, the solution wasconcentrated, dissolved in 1:1 acetic acid: water+3004 TFA (7 mL) andpurified by flash chromatography (100 g column, 5-65 ACN/water), toprovide the TFA salt of the title compound (165 mg, 0.293 mmol, 30%yield). (m/z): [M+H]⁺ calcd for C₂₄H₂₄FN₅O₃ 450.19 found 450.

Example 2:(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone

To a solution of(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, TFA (179 mg, 0.310 mmol), N,N-dimethylazetidin-3-amine, 2 HCl (107mg, 0.465 mmol), and DIPEA (0.162 mL 0.930 mmol) in DMF (4 mL), wasadded HATU (177 mg, 0.465 mmol) and the reaction mixture was stirred atRT overnight. Hydrazine (5 eq) was added, the reaction mixture wasconcentrated and purified by preparative HPLC to provide the TFA salt ofthe title compound (63 mg, 26% yield). (m/z): [M+H]⁺ calcd forC₃₀H₃₆FN₇O₂ 546.29 found 546.7. ¹H NMR (400 MHz, DMSO-d₆) δ 9.90 (s,1H), 8.29 (dd, 1H), 7.34 (s, 1H), 7.07 (d, 1H), 7.01 (d, 1H), 6.89 (d,1H), 4.35-4.18 (m, 1H), 4.11-3.94 (m, 1H), 3.94-3.73 (m, 3H), 3.70-3.57(m, 2H), 3.06-2.94 (m, 2H), 2.87-2.66 (m, 2H), 2.48-2.40 (m, 2H),2.13-2.00 (m, 6H), 1.07 (t, 3H), 1.03-0.93 (m, 6H).

Example 4:(S)-(3-(dimethylamino)azetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone

To a solution of(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, TFA (30 mg, 0.052 mmol), N,N-dimethylazetidin-3-amine, 2HCl (27.0mg, 0.156 mmol), and DIPEA (0.064 mL, 0.364 mmol) in DMF (1.5 mL), wasadded HATU (29.6 mg, 0.078 mmol) and the reaction mixture was stirred atRT overnight. Hydrazine (5 eq) was added, the reaction mixture wasstirred at RT for 10 min, concentrated and purified by preparative HPLCto provide the TFA salt of the title compound (29.6 mg, 74% yield).(m/z): [M+H]⁺ calcd for C₃₀H₃₆FN₇O₂ 546.29 found 546.6.

Example 8:(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone

To a solution of(S)-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, TFA (30 mg, 0.052 mmol), N,N-3-trimethylazetidin-3-amine, 2 HCl(29.2 mg, 0.156 mmol), and DIPEA (0.073 mL, 0.416 mmol) in DMF (1 mL),was added HATU (29.6 mg, 0.078 mmol) and the reaction mixture wasstirred at RT overnight. Hydrazine (5 eq) was added, the reactionmixture was concentrated and purified by preparative HPLC to provide theTFA salt of the title compound (24.7 mg, 60% yield). (m/z): [M+H]⁺ calcdfor C₃₁H₃₈FN₇O₂ 560.31 found 560.2.

Example 8-22:(S)-(3-(dimethylamino)azetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone

(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, TFA (30 mg, 0.053 mmol), N,N-dimethylazetidin-3-amine (16 mg, 0.16mmol), and DIPEA (0.037 mL, 0.213 mmol) were dissolved in DMF (1.0 ml),then HATU (30.4 mg, 0.080 mmol) was added and the reaction mixture wasstirred at room temperature for 6 hours. Hydrazine (15 μL) was addedthen the solution was concentrated and purified by preparative HPLC toprovide the TFA salt of the title couple (27 mg, 66% yield). (m/z):[M+H]⁺ calcd for C₂₉H₃₄FN₇O₂ 532.6 found 532.2.

Example 8-23:(S)-(3-(dimethylamino)-3-methylazetidin-1-yl)(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)methanone

(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, TFA (30 mg, 0.053 mmol), N,N,3-trimethylazetidin-3-amine (18 mg,0.16 mmol), and DIPEA (0.037 mL, 0.213 mmol) were dissolved in DMF (1.0ml), then HATU (30.4 mg, 0.080 mmol) was added and the reaction mixturewas stirred at room temperature for 6 hours. Hydrazine (15 μL) was addedthen the solution was concentrated and purified by preparative HPLC toprovide the TFA salt of the title couple (28 mg, 68% yield). (m/z):[M+H]⁺ calcd for C₃₀H₃₆FN₇O₂ 546.7 found 546.2.

Example 8-14:(S)-(5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-6-yl)(3-(piperidin-1-yl)azetidin-1-yl)methanone,2TFA

(S)-5-ethyl-2-(6-(2-ethyl-5-fluoro-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylicacid, TFA (40 mg, 0.071 mmol), 1-(3-azetidinyl)piperidine (29.9 mg,0.213 mmol), and DIPEA (0.050 ml, 0.284 mmol) were dissolved in DMF (1.5ml), then HATU (40.5 mg, 0.106 mmol) was added and the reaction mixturewas stirred at room temperature for 2 hours. Hydrazine (0.011 ml, 0.355mmol) was added and the reaction mixture was stirred at room temperaturefor 10 minutes. The solution was then concentrated purified bypreparative HPLC to provide the TFA salt of the title compound (36 mg,63% yield). (m/z): [M+H]+ calcd for C₃₂H₃₈FN₇O₂ 572.7 found 572.5.

Using similar synthetic methods, the compounds of Table 1 were prepared.In the following tables, a blank in any column indicates a hydrogenatom, a * in a structure heading a table indicates a chiral center, andthe notation (R) or (S) in front of a substituent denotes theconfiguration of the carbon atom to which the substituent is attached.

TABLE 1

Calc Found Ex. No. * R¹ R⁵ R⁶ R⁷ Formula [M + H]⁺ [M + H]⁺ 8-1  CH₃—(CH₂)₅— C₃₁H₃₆FN₇O₂ 558.29 557.7 8-2  CH₃ CH₃ CH₃ C₂₈H₃₂FN₇O₂ 518.26517.8 8-3  nPr CH₃ C₃₀H₃₆FN₇O₂ 546.29 546.2 8-4  C₂H₅ —(CH₂)₅—C₃₂H₃₈FN₇O₂ 572.31 571.7 8-5  nPr —(CH₂)₅— C₃₃H₄₀FN₇O₂ 586.32 585.7 8-6 iPr —(CH₂)₅— C₃₃H₄₀FN₇O₂ 586.32 585.9 8-7  iPr CH₃ CH₃ C₃₀H₃₆FN₇O₂546.29 546.2 8-8  C₂H₅ CH₃ CH₃ C₂₉H₃₄FN₇O₂ 532.28 532.2 8-9  CH₃ CH₃ CH₃CH₃ C₂₉H₃₄FN₇O₂ 532.28 532.2 8-10 C₂H₅ CH₃ CH₃ CH₃ C₃₀H₃₆FN₇O₂ 546.29546.2 8-11 nPr CH₃ CH₃ CH₃ C₃₁H₃₈FN₇O₂ 560.31 560.2 8-12 iPr CH₃ CH₃ CH₃C₃₁H₃₈FN₇O₂ 560.31 560.3 8-13 (R) nPr CH₃ CH₃ CH₃ C₃₀H₃₆FN₇O₂ 546.29546.5 8-14 (S) C₂H₅ —(CH₂)₅— C₃₂H₃₈FN₇O₂ 572.31 572.3 8-15 (R) C₂H₅—(CH₂)₅— C₃₂H₃₈FN₇O₂ 572.31 572.2 8-16 (S) iPr CH₃ CH₃ CH₃ C₃₁H₃₈FN₇O₂560.31 560.2 8-17 (R) iPr CH₃ CH₃ C₃₀H₃₆FN₇O₂ 546.29 546.2 8-18 (R) iPrCH₃ CH₃ CH₃ C₃₁H₃₈FN₇O₂ 560.31 560.2 8-19 (R) nPr CH₃ CH₃ CH₃C₃₁H₃₈FN₇O₂ 560.31 560.6 8-20 (R) C₂H₅ CH₃ CH₃ C₂₉H₃₄FN₇O₂ 532.28 532.28-21 (R) C₂H₅ CH₃ CH₃ CH₃ C₃₀H₃₆FN₇O₂ 546.29 546.2 8-22 (S) C₂H₅ CH₃ CH₃C₂₉H₃₄FN₇O₂ 532.28 532.2 8-23 (S) C₂H₅ CH₃ CH₃ CH₃ C₃₀H₃₆FN₇O₂ 546.29546.2Biological Assays

The compounds of the invention have been characterized in one or more ofthe following biological assays.

Assay 1: Biochemical JAK Kinase Assays

A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 andTyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH7.5, 0.01% Brij-35, 10 mM MgCl₂, and 1 mM EGTA). Recombinant GST-taggedJAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained fromLife Technologies.

Serially diluted compounds were pre-incubated with each of the four JAKenzymes and the substrate in white 384-well microplates (Corning) atambient temperature for 1 h. ATP was subsequently added to initiate thekinase reactions in 10 μL total volume, with 1% DMSO. The final enzymeconcentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and0.25 nM respectively; the corresponding Km ATP concentrations used are25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200nM for all four assays. Kinase reactions were allowed to proceed for 1hour at ambient temperature before a 10 μL preparation of EDTA (10 mMfinal concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (LifeTechnologies, 2 nM final concentration) in TR-FRET dilution buffer (LifeTechnologies) was added. The plates were allowed to incubate at ambienttemperature for 1 h before being read on the EnVision reader (PerkinElmer). Emission ratio signals (520 nm/495 nm) were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software (GraphPadSoftware). Results were expressed as pIC₅₀ (negative logarithm of IC₅₀)and subsequently converted to pK_(i) (negative logarithm of dissociationconstant, Ki) using the Cheng-Prusoff equation.

Test compounds having a lower K_(i) value or higher pK_(i) value in eachof the four JAK assays show greater inhibition of JAK activity.

Assay 2: Cellular JAM Potency Assay

The AlphaScreen JAM cellular potency assay was carried out by measuringinterleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation inBEAS-2B human lung epithelial cells (ATCC). The anti-STAT6 antibody(Cell Signaling Technologies) was conjugated to AlphaScreen acceptorbeads (Perkin Elmer), while the anti-pSTAT6 (pTyr641) antibody (CellSignaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin(Thermo Scientific).

BEAS-2B cells were grown at 37° C. in a 5% CO₂ humidified incubator in50% DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS(Hyclone), 100 U/mL penicillin, 100 μg/mL streptomycin (LifeTechnologies), and 2 mM GlutaMAX (Life Technologies). On day 1 of theassay, cells were seeded at a 7,500 cells/well density in whitepoly-D-lysine-coated 384-well plates (Corning) with 254 medium, and wereallowed to adhere overnight in the incubator. On day 2 of the assay, themedium was removed and replaced with 12 μL of assay buffer (Hank'sBalanced Salt Solution/HBSS, 25 mM HEPES, and 1 mg/ml bovine serumalbumin/BSA) containing dose-responses of test compounds. Compounds wereserially diluted in DMSO and then diluted another 1000-fold in media tobring the final DMSO concentration to 0.1%. Cells were incubated withtest compounds at 37° C. for 1 h, and followed by the addition of 12 μlof pre-warmed IL-13 (80 ng/mL in assay buffer) for stimulation. Afterincubating at 37° C. for 30 min, the assay buffer (containing compoundand IL-13) was removed, and 10 μL of cell lysis buffer (25 mM HEPES,0.1% SDS, 1% NP-40, 5 mM MgCl₂, 1.3 mM EDTA, 1 mM EGTA, and supplementwith Complete Ultra mini protease inhibitors and PhosSTOP from RocheDiagnostics). The plates were shaken at ambient temperature for 30 minbefore the addition of detection reagents. A mixture ofbiotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beads was addedfirst and incubated at ambient temperature for 2 h, followed by theaddition of streptavidin conjugated donor beads (Perkin Elmer). After aminimum of 2 h incubation, the assay plates were read on the EnVisionplate reader. AlphaScreen luminescence signals were recorded andutilized to calculate the percent inhibition values based on DMSO andbackground controls.

For dose-response analysis, percent inhibition data were plotted vs.compound concentrations, and IC₅₀ values were determined from a4-parameter robust fit model with the Prism software. Results may alsobe expressed as the negative logarithm of the IC₅₀ value, pIC₅₀.

Test compounds having a lower IC₅₀ value or higher pIC₅₀ value in thisassay show greater inhibition of IL-13 induced STAT6 phosphorylation.

In Vitro Assay Results

Selected compounds of the invention were tested in the four JAK enzymeassays; JAK1, JAK2, JAK3, and Tyk2, and the BEAS-2B cellular potencyassay described above. As shown in Table 19 below, it was observed thatJAK1 enzyme potency was predictive of both pan-JAK enzyme activity andcellular potency in the BEAS-2B assay. Therefore, all of the compoundsmade were tested in the JAK1 enzyme assay and the BEAS-2B cellular assayand the great majority were also tested in the JAK3 enzyme assay. All ofthe compounds exhibited JAK1 K_(i) values between 0.04 nM and 0.6 nM(pK_(i) between 9.2 and 10.4). The compounds tested in the JAK3 enzymeassay exhibited K_(i) values between 0.08 nM and 0.5 nM (pK_(i) between9.3 and 10.1). The compounds tested exhibited IC₅₀ values in the BEAS-2Bassay between 3 nM and 100 nM (pIC₅₀ between 7 and 8.5).

TABLE 2 JAK1 JAK2 JAK3 Tyk2 BEAS-2B Example K_(i) K_(i) K_(i) K_(i) IC₅₀Number (nM) (nM) (nM) (nM) (nM) 2 0.04 0.02 0.10 0.32 4.1 4 0.06 0.137.4 8 0.06 0.16 8.5 8-1 0.06 0.18 6.3 8-2 0.06 0.25 12.6 8-3 0.06 0.165.0 8-4 0.06 0.13 10.0 8-5 0.08 0.20 25.1 8-6 0.06 0.25 15.8 8-7 0.060.20 3.1 8-8 0.08 0.28 5.0 8-9 0.05 0.16 7.1 8-10 0.08 0.32 3.2 8-110.10 0.32 6.3 8-12 0.06 0.25 4.0 8-13 0.1 0.32 4.0 8-14 0.08 0.20 12.68-15 0.08 0.03 0.40 0.40 5.6 8-16 0.10 0.32 5.0 8-17 0.05 0.13 8.0 8-180.10 0.32 4.0 8-19 0.06 0.20 8.0 8-20 0.05 0.20 5.0 8-21 0.05 0.25 4.08-22 0.06 0.32 5.25 8-23 0.05 0.2 7.7

Assay 3: Pharmacokinetics in Plasma and Lung in Mouse

Plasma and lung levels of test compounds and ratios thereof weredetermined in the following manner. BALB/c mice from Charles RiverLaboratories were used in the assay. Test compounds were individuallyformulated in 20% propylene glycol in pH 4 citrate buffer at aconcentration of 0.2 mg/mL and 50 μL of the dosing solution wasintroduced into the trachea of a mouse by oral aspiration. At varioustime points (typically 0.167, 2, 6, 24 hr) post dosing, blood sampleswere removed via cardiac puncture and intact lungs were excised from themice. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4minutes at approximately 12,000 rpm at 4° C. to collect plasma. Lungswere padded dry, weighed, and homogenized at a dilution of 1:3 insterile water. Plasma and lung levels of test compound were determinedby LC-MS analysis against analytical standards constructed into astandard curve in the test matrix. A lung to plasma ratio was determinedas the ratio of the lung AUC in μg hr/g to the plasma AUC in μg hr/mL,where AUC is conventionally defined as the area under the curve of testcompound concentration vs. time. Compounds of the invention exhibitedexposure in lung from one to two orders of magnitude greater thanexposure in plasma in mouse. All of the compounds profiled in this assayexhibited a half-life between about 4.5 and about 14 hours.

Assay 4: Murine (Mouse) Model of IL-13 Induced pSTAT6 Induction in LungTissue

Il-13 is an important cytokine underlying the pathophysiology of asthma(Kudlacz et al. Eur. J. Pharmacol, 2008, 582,154-161). IL-13 binds tocell surface receptors activating members of the Janus family of kinases(JAK) which then phosphorylate STAT6 and subsequently activates furthertranscription pathways. In the described model, a dose of IL-13 wasdelivered locally into the lungs of mice to induce the phosphorylationof STAT6 (pSTAT6) which is then measured as the endpoint.

Adult balb/c mice from Harlan were used in the assay. On the day ofstudy, animals were lightly anesthetized with isoflurane andadministered either vehicle or test compound (1 mg/mL, 50 μL totalvolume over several breaths) via oral aspiration. Animals were placed inlateral recumbency post dose and monitored for full recovery fromanesthesia before being returned to their home cage. Four hours later,animals were once again briefly anesthetized and challenged with eithervehicle or IL-13 (0.03 μg total dose delivered, 50 μL total volume) viaoral aspiration before being monitored for recovery from anesthesia andreturned to their home cage. One hour after vehicle or IL-13administration, lungs were collected for both pSTAT6 detection using ananti-pSTAT6 ELISA (rabbit mAb capture/coating antibody; mouse mAbdetection/report antibody: anti-pSTAT6-pY641; secondary antibody:anti-mouse IgG-HRP) and analyzed for total drug concentration asdescribed above in Assay 3.

Selected compounds of the invention were tested in the assay. Activityin the model is evidenced by a decrease in the level of pSTAT6 presentin the lungs of treated animals at 5 hours compared to the vehicletreated, IL-13 challenged control animals. The difference between thecontrol animals which were vehicle-treated, IL-13 challenged and and thecontrol animals which were vehicle-treated, vehicle challenged dictatedthe 0% and 100% inhibitory effect, respectively, in any givenexperiment. Exemplary compounds of the invention were tested in theassay, and exhibited inhibition of STAT6 phosphorylation at 4 hoursafter IL-13 challenge as documented below.

Confirming the relevance of the JAK-STAT pathway in airway inflammation,compounds which have demonstrated in vivo target engagement in theIL13-induced pSTAT6 mouse model are subsequently tested and proven to beefficacious in a mouse model of allergen-induced eosinophilicinflammation.

In Vivo Assay Results

Selected compounds of the invention were characterized in both thepharmacokinetic assay (Assay 3) and pharmacodynamic assay (Assay 4). Agood correlation was observed between test compound concentration inlung determined in the pharmacokinetic assay and in the pharmacodynamicassay at a similar time points post dosing. Observation of significantcompound concentration in the mouse lung in the pharmacodynamic assayconfirmed that the observed inhibition of IL-13 induced pSTAT6 inductionwas a result of the activity of the test compound.

In the following table, for the ratio of lung exposure to plasmaexposure (Assay 3), A denotes a ratio 100-200, B denotes a ratio between50 and 100, and C denotes a ratio between 20 and 50. For the percentinhibition of IL-13 induced pSTAT6 induction (Assay 4), A representsbetween 60% and 80% inhibition, B represents between 40% and 60%inhibition and C represents between 25% and 40% inhibition.

TABLE 3 Lung to Plasma pSTAT6 Example ratio inhibition Number Assay 3Assay 4 2 A B 4 C A 8 B B 8-3 C B 8-7 C B 8-13 B 8-15 C 8-16 B C 8-22 BB 8-23 B B

Assay 5: Murine Model of Alternaria alternata-Induced EosinophilicInflammation of the Lung

Airway eosinophilia is a hallmark of human asthma. Alternaria alternatais a fungal aeroallergen that can exacerbate asthma in humans andinduces eosinophilic inflammation in the lungs of mice (Havaux et al.Clin Exp Immunol. 2005 February; 139(2):179-88). In mice, it has beendemonstrated that alternaria indirectly activates tissue resident type 2innate lymphoid cells in the lung, which respond to (e.g. IL-2 and IL-7)and release JAK-dependent cytokines (e.g. IL-5 and IL-13) and coordinateeosinophilic inflammation (Bartemes et al. J Immunol. 2012 Feb. 1;188(3):1503-13).

Seven- to nine-week old male C57 mice from Taconic were used in thestudy. On the day of study, animals were lightly anesthetized withisoflurane and administered either vehicle or test compound (0.03-1.0mg/mL, 50 μL total volume over several breaths) via oropharyngealaspiration. Animals were placed in lateral recumbency post dose andmonitored for full recovery from anesthesia before being returned totheir home cage. One hour later, animals were once again brieflyanesthetized and challenged with either vehicle or alternaria extract(200 ug total extract delivered, 50 μL total volume) via oropharyngealaspiration before being monitored for recovery from anesthesia andreturned to their home cage. Forty-eight hours after alternariaadministration, bronchoalveolar lavage fluid (BALF) was collected andeosinophils were counted in the BALF using the Advia 120 HematologySystem (Siemens).

Selected compounds of the invention demonstrating in vivo activity inthe IL-13-pSTAT6 pharmacodynamic assay were tested in this alternariaassay. Activity in the model is evidenced by a decrease in the level ofeosinophils present in the BALF of treated animals at forty-eight hourscompared to the vehicle treated, alternaria challenged control animals.Data are expressed as percent inhibition of the vehicle treated,alternaria challenged BALF eosinophils response. To calculate percentinhibition, the number of BALF eosinophils for each condition isconverted to percent of the average vehicle treated, alternariachallenged BALF eosinophils and subtracted from one-hundred percent.Exemplary compounds of the invention were tested in the assay andexhibited inhibition of BALF eosinophil counts at forty-eight hoursafter alternaria challenge as documented below.

In Vivo Assay Results

All of the compounds tested demonstrated a range of inhibition (73%-93%)of alternaria-induced BALF eosinophils. The following table reflects themaximum statistically significant percent inhibition of the vehicletreated, alternaria challenged level of eosinophil induction.

TABLE 4 Percent Inhibition of Alternaria-induced Example Number BALFEosinophils 2 74 4 79

Assay 6: IL-5 Mediated Eosinophil Survival Assay

The potency of the test compound for IL-5 mediated eosinophil survivalwas measured in human eosinophils isolated from human whole blood(AllCells). Because IL-5 signals through JAK, this assay provides ameasure of JAK cellular potency.

Human eosinophils were isolated from fresh human whole blood (AllCells)of healthy donors. Blood was mixed with 4.5% Dextran (Sigma-Aldrich) ina 0.9% sodium chloride solution (Sigma-Aldrich). Red blood cells wereleft to sediment for 35 minutes. The leukocyte rich upper layer wasremoved and layered over Ficoll-Paque (GE Healthcare) and centrifuged at600 g for 30 minutes. The plasma and mononuclear cell layers wereremoved before the granulocyte layer was lysed with water to remove anycontaminating red blood cells. Eosinophils were further purified using ahuman eosinophil isolation kit (Miltenyi Biotec). A fraction of thepurified eosinophils were incubated with anti-CD16 FITC (MiltenyiBiotec) for 10 minutes at 4° C. in the dark. Purity was analyzed using aLSRII flow cytometer (BD Biosciences).

Cells were cultured in a 37° C., 5% CO₂ humidified incubator in RPMI1640 (Life Technologies) supplemented with 10% Heat Inactivated FetalBovine Serum (FBS, Life Technologies), 2 mM Glutamax (LifeTechnologies), 25 mM HEPES (Life Technologies) and 1× Pen/Strep (LifeTechnologies). Cells were seeded at 10,000 cells/well in media (50 μL).The plate was centrifuged at 300 g for 5 minutes and supernatantsremoved. Compounds were serially diluted in DMSO and then dilutedanother 500-fold to a 2× final assay concentration in media. Testcompounds (50 μL/well) were added to cells, and incubated at 37° C., 5%CO₂ for 1 hour, followed by the addition of IL-5 (R&D Systems; finalconcentrations 1 ng/mL and 10 pg/ml) in pre-warmed assay media (50 μL)for 72 hours.

After cytokine stimulation, cells were centrifuged at 300 g for 5 minand washed twice with cold DPBS (Life Technologies). To access viabilityand apoptosis, cells were incubated with Propidium Iodide (Thermo FisherScientific) and APC Annexin V (BD Biosciences) and analyzed using aLSRII flow cytometer (BD Biosciences). IC₅₀ values were determined fromanalysis of the viability curves of percent cell viability vs compoundconcentration. Data are expressed as pIC₅₀ (negative decadic logarithmIC₅₀) values. The compound of example 2 exhibited a pIC₅₀ value of7.6±0.5 in the presence of 10 pg/ml IL-5 and a pIC₅₀ value of 6.2±0.1 inthe presence of 1 ng/ml IL-5.

Assay 7: Inhibition of IFNγ and IL-27 Induced Chemokines CXCL9 andCXCL10 in Human 3D Airway Cultures

EpiAirway tissue cultures were obtained from Mattek (AIR-100). Cultureswere derived from asthmatic donors. In a cell culture insert, humanderived tracheal/bronchial epithelial cells were grown anddifferentiated on a porous membrane support, allowing an air-liquidinterface with warmed culture medium below the cells and a gaseous testatmosphere above. Tissues were cultured in maintenance media (Mattek,AIR-100-MM) in a 37° C., 5% CO2 humidified incubator. Four donors weretested. On Day 0, tissue cultures were treated with test compounds at 10μM, 1 μM and/or 0.1 μM. Compounds were diluted in dimethyl sulfoxide(DMSO, Sigma) to a final concentration of 0.1%. DMSO at 0.1% was used asa vehicle control. Test compounds were incubated with cultures for 1hour at 37° C., 5% CO₂, followed by the addition of pre-warmed mediacontaining IFNγ (R&D Systems) or IL-27 (R&D Systems) at a finalconcentration at 100 ng/ml. Tissue cultures were maintained for 8 days.Media was replaced every 2 days with fresh media containing compoundsand IFNγ or IL-27. On Day 8, tissue cultures and supernatants werecollected for analysis. Supernatant samples were assayed for CXCL10(IP-10) and CXCL9 (MIG) using luminex analysis (EMD Millipore). Data isexpressed as % Inhibition+/−standard deviation (±STDV). Percentinhibition was determined by compound inhibitory potency against IFNγ orIL-27 induced CXCL10 or CXCL9 secretion compared to vehicle treatedcells. Data is the average from 3 or 4 donors. The compound of example 2was able to inhibit IFNγ induced CXCL10 secretion by 101%±2.0 (at 10μM), 65%±29 (at 0.1 μM) and 6%±11 (at 0.1 μM) when compared to vehiclecontrol. The compound of example 2 was able to inhibit IFNγ inducedCXCL9 secretion by 93%±13 (at 10 μM) and 24%±49 (at 1 μM) when comparedto vehicle. The compound of example 2 was able to inhibit IL-27 inducedCXCL10 secretion by 108%±11 (at 10 μM), 101%±6 (at 0.1 μM) and 69%±10(at 0.1 μM) when compared to vehicle control. The compound of example 2was able to inhibit IL-27 induced CXCL9 secretion by 100%±0 (at 10 μM),97%±3.6 (at 0.1 μM) and 57%±28 (at 0.1 μM) when compared to vehiclecontrol.

Assay 8: Cellular JAK Potency Assay: Inhibition of IL-2/Anti-CD3Stimulated IFNγ in Human PBMCs

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated interferon gamma (IFNγ) was measured in humanperipheral blood mononuclear cells (PBMCs) isolated from human wholeblood (Stanford Blood Center). Because IL-2 signals through JAK, thisassay provides a measure of JAK cellular potency.

(1) Human peripheral blood mononuclear cells (PBMC) were isolated fromhuman whole blood of healthy donors using a ficoll gradient. Cells werecultured in a 37° C., 5% CO₂ humidified incubator in RPMI (LifeTechnologies) supplemented with 10% Heat Inactivated Fetal Bovine Serum(FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES(Life Technologies) and 1× Pen/Strep (Life Technologies). Cells wereseeded at 200,000 cells/well in media (50 μL) and cultured for 1 h.Compounds were serially diluted in DMSO and then diluted another500-fold (to a 2× final assay concentration) in media. Test compounds(100 μL/well) were added to cells, and incubated at 37° C., 5% CO₂ for 1h, followed by the addition of IL-2 (R&D Systems; final concentration100 ng/mL) and anti-CD3 (BD Biosciences; final concentration 1 μg/mL) inpre-warmed assay media (50 μL) for 24 h.

(2) After cytokine stimulation, cells were centrifuged at 500 g for 5min and supernatants removed and frozen at −80° C. To determine theinhibitory potency of the test compound in response to IL-2/anti-CD3,supernatant IFNγ concentrations were measured via ELISA (R&D Systems).IC₅₀ values were determined from analysis of the inhibition curves ofconcentration of IFNγ vs compound concentration. Data are expressed aspIC₅₀ (negative decadic logarithm IC₅₀) values. The compound of Example2 exhibited a pIC₅₀ value of about 7.1 in this assay.

Assay 9: Cellular JAK Potency Assay: Inhibition of IL-2 StimulatedpSTAT5 in CD4+ T Cells

The potency of the test compound for inhibition of interleukin-2(IL-2)/anti-CD3 stimulated STATS phosphorylation was measured inCD4-positive (CD4+) T cells in human peripheral blood mononuclear cells(PBMCs) isolated from human whole blood (Stanford Blood Center) usingflow cytometry. Because IL-2 signals through JAK, this assay provides ameasure of JAK cellular potency.

CD4+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD4 antibody (Clone RPA-T4, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT5 antibody (pY694, Clone 47, BD Biosciences)was used to detect STATS phosphorylation.

(1) The protocol of Assay 8 paragraph (1) was followed with theexception that the cytokine stimulation with anti-CD3 was performed for30 min instead of 24 h.

(2) After cytokine stimulation, cells were fixed with pre warmed fixsolution (200 μL; BD Biosciences) for 10 min at 37° C., 5% CO₂, washedtwice with DPBS buffer (1 mL, Life Technologies), and resuspended in icecold Perm Buffer III (1000 μL, BD Biosciences) for 30 min at 4° C. Cellswere washed twice with 2% FBS in DPBS (FACS buffer), and thenresuspended in FACS buffer (100 μL) containing anti-CD4 PE (1:50dilution) and anti-CD3 anti-CD3Alexa Fluor 647 (1:5 dilution) for 60 minat room temperature in the dark. After incubation, cells were washedtwice in FACS buffer before being analyzed using a LSRII flow cytometer(BD Biosciences). To determine the inhibitory potency of test compoundsin response to IL-2/anti-CD3, the median fluorescent intensity (MFI) ofpSTAT5 was measured in CD4+ T cells. IC₅₀ values were determined fromanalysis of the inhibition curves of MFI vs compound concentration. Dataare expressed as pIC₅₀ (negative decadic logarithm IC₅₀) values. Thecompound of Example 2 exhibited a pIC₅₀ value of about 7.3 in thisassay.

Assay 10: Cellular JAK Potency Assay: Inhibition of IL-4 StimulatedpSTAT6 in CD3+ T Cells

The potency of the test compound for inhibition of interleukin-4 (IL-4)stimulated STAT6 phosphorylation was measured in CD3-positive (CD3+) Tcells in human peripheral blood mononuclear cells (PBMCs) isolated fromhuman whole blood (Stanford Blood Center) using flow cytometry. BecauseIL-4 signals through JAK, this assay provides a measure of JAK cellularpotency.

CD3+ T cells were identified using a phycoerythrobilin (PE) conjugatedanti-CD3 antibody (Clone UCHT1, BD Biosciences), while an Alexa Fluor647 conjugated anti-pSTAT6 antibody (pY641, Clone 18/P, BD Biosciences)was used to detect STAT6 phosphorylation.

Human peripheral blood mononuclear cells (PBMC) were isolated from humanwhole blood of healthy donors as in Assays 8 and 9. Cells were seeded at250,000 cells/well in media (200 μL), cultured for 1 h and thenresuspended in assay media (50 μL) (RPMI supplemented with 0.1% bovineserum albumin (Sigma), 2 mM Glutamax, 25 mM HEPES and 1× Penstrep)containing various concentrations of test compounds. Compounds wereserially diluted in DMSO and then diluted another 500-fold (to a 2×final assay concentration) in assay media. Test compounds (50 μL) wereincubated with cells at 37° C., 5% CO₂ for 1 h, followed by the additionof IL-4 (50 μL) (R&D Systems; final concentration 20 ng/mL) inpre-warmed assay media for 30 min. After cytokine stimulation, cellswere fixed with pre-warmed fix solution (100 μL) (BD Biosciences) for 10min at 37° C., 5% CO₂, washed twice with FACS buffer (1 mL) (2% FBS inDPBS), and resuspended in ice cold Perm Buffer III (1000 μL) (BDBiosciences) for 30 min at 4° C. Cells were washed twice with FACSbuffer, and then resuspended in FACS buffer (100 μL) containing anti-CD3PE (1:50 dilution) and anti-pSTAT6 Alexa Fluor 647 (1:5 dilution) for 60min at room temperature in the dark. After incubation, cells were washedtwice in FACS buffer before being analyzed using a LSRII flow cytometer(BD Biosciences).

To determine the inhibitory potency of the test compound in response toIL-4, the median fluorescent intensity (MFI) of pSTAT6 was measured inCD3+ T cells. IC₅₀ values were determined from analysis of theinhibition curves of MFI vs compound concentration. Data are expressedas pIC₅₀ (negative decadic logarithm IC₅₀). The compound of Example 2exhibited a pIC₅₀ value of 7.9 in this assay.

Assay 11: Cellular JAK Potency Assay: Inhibition of IL-6 StimulatedpSTAT3 in CD3+ T Cells

A protocol analogous to that of Assay 10 was used to determine thepotency of the test compound for inhibition of interleuken-6 (IL-6)stimulated STAT3 phosphorylation. An Alexa Fluor 647 conjugatedanti-pSTAT3 antibody (pY705, Clone 4/P, BD Biosciences) was used todetect STAT3 phosphorylation.

The compound of Example 2 exhibited a pIC₅₀ value of 7.2 in this assay.

Crystal Structure

A co-crystal structure was obtained of the compound of Example 2 boundto human JAK1 at a resolution of 2.28 Å. The ligand was observed to bindin the ATP binding site. Seven specific hydrogen bonding interactionswere identified based upon a distance of 3.5 Å or less between donor andacceptor atoms. Of particular note, a hydrogen bonding interaction wasidentified between the carbonyl of the exocyclic amide of the compoundof Example 2 and the sidechain of Arg879 of JAK1. In earlier modelingstudies this interaction had been proposed as a way to provideselectivity for JAK1 over other tyrosine kinases, as otherwise closelyrelated kinases (e.g. TRKA, VEGFR, ABL1) did not possess an arginineresidue at the equivalent location. The observed results of the hydrogenbonding interaction in the crystal structure and improved kinomeselectivity compared to series not possessing the exocyclic amidevalidate this design hypothesis.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A pharmaceutical composition comprising acompound of formula:

or a pharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier.
 2. The pharmaceutical compositionof claim 1, wherein the pharmaceutical composition is suitable forinhaled administration.
 3. The pharmaceutical composition of claim 1,wherein the pharmaceutical composition is suitable for nebulizedadministration.
 4. The pharmaceutical composition of claim 1, whereinthe composition is administered with an inhaler delivery device.
 5. Thepharmaceutical composition of claim 4, wherein the inhaler deliverydevice is selected from the group consisting of a dry powder inhaler, ametered-dose inhaler, and a nebulizer inhaler.
 6. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition is in theform of a solution.
 7. The pharmaceutical composition of claim 1,wherein the pharmaceutical composition is in the form of a dry powder.8. The pharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises lactose, starch, mannitol, dextrose, polylacticacid, or polylactide-co-glycolide or a combination thereof.
 9. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition comprises glucose, or sorbitol, or a combination thereof.10. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition comprises one or more other therapeuticagents.
 11. The pharmaceutical composition of claim 10, wherein the oneor more therapeutic agents are selected from the group consisting of abeta 2 adrenoceptor agonist, a muscarinic receptor antagonist, aglucocorticoid agonist, and a leukotriene antagonist.
 12. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition contains from about 0.01 to about 95% by weight of thecompound or a pharmaceutically-acceptable salt thereof.
 13. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition contains from about 0.05 to about 30% by weight of thecompound or a pharmaceutically-acceptable salt thereof.
 14. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition contains from about 0.1% to about 10% by weight of thecompound or a pharmaceutically-acceptable salt thereof.
 15. Thepharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is loaded into a dry powder dispenser, or into inhalationcartridges or capsules for use with a dry powder delivery device. 16.The pharmaceutical composition of claim 1, wherein the pharmaceuticalcomposition is a solution or suspension comprising from about 0.05 μg/mLto about 20 mg/mL of the compound and has a pH of about 3 to about 8.17. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is a dry powder comprising about 0.1 mg toabout 4 mg of the compound.
 18. The pharmaceutical composition of claim1, wherein the pharmaceutical composition is loaded into individualblisters of a peelable blister pack.